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Enrichment and characterization of phosphopeptides by immobilized metal affinity chromatography (IMAC) and mass spectrometry
DEFF Research Database (Denmark)
Thingholm, Tine E; Jensen, Ole N
The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and this...... phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10-11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures....
Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns
Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that ...
Cromatografia de afinidade por íons metálicos imobilizados (IMAC) de biomoléculas: aspectos fundamentais e aplicações tecnológicas Immobilized metal-ion affinity chromatography (IMAC) of biomolecules: fundamental aspects and technological applications
Igor Tadeu Lazzarotto Bresolin; Everson Alves Miranda; Sônia Maria Alves Bueno
Immobilized Metal Ion Affinity Cromatography - IMAC - is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be design...
Cromatografia de afinidade por íons metálicos imobilizados (IMAC de biomoléculas: aspectos fundamentais e aplicações tecnológicas Immobilized metal-ion affinity chromatography (IMAC of biomolecules: fundamental aspects and technological applications
Directory of Open Access Journals (Sweden)
Igor Tadeu Lazzarotto Bresolin
Full Text Available Immobilized Metal Ion Affinity Cromatography - IMAC - is a group-specific based adsorption applied to the purification and structure-function studies of proteins and nucleic acids. The adsorption is based on coordination between a metal ion chelated on the surface of a solid matrix and electron donor groups at the surface of the biomolecule. IMAC is a highly selective, low cost, and easily scaled-up technique being used in research and commercial operations. A separation process can be designed for a specific molecule by just selecting an appropriate metal ion, chelating agent, and operational conditions such as pH, ionic strength, and buffer type.
Phosphopeptide enrichment by immobilized metal affinity chromatography
DEFF Research Database (Denmark)
Thingholm, Tine E.; Larsen, Martin R.
binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10–11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe 3+ for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide......Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...... charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from...
Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.
Swaney, Danielle L; Villén, Judit
Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved. PMID:26933247
Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.
Thingholm, Tine E; Larsen, Martin R
Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922
Institute of Scientific and Technical Information of China (English)
AanTianwei; XuWeijiang; 等
A new and an inexpensive adsorbent of chitosan coated silica for immobilized metal affinity chromatography(IMAC) was studied.After a double coating,the chitosan coated on silica beads could be up to 53.4mg/g silica beads.When pH>3.8,the metal ligand Cu2+ was chelated on the coated chitosan with a bound capacity of 14.6mg/g chitosan without introducing iminodiacetic acid(IDA).
Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins : a wash step with a low concentration of EDTA
Westra, DF; Welling, GW; Koedijk, DGAM; Scheffer, AJ; The, TH; Welling-Wester, S
Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for t
Removal of PCR error products and unincorporated primers by metal-chelate affinity chromatography.
Directory of Open Access Journals (Sweden)
Indhu Kanakaraj
Full Text Available Immobilized Metal Affinity Chromatography (IMAC has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and "histidine tags" genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu(2+-iminodiacetic acid (IDA agarose spin column, 94-99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu(2+-IDA agarose can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs.
Large-scale analysis of in Vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry
DEFF Research Database (Denmark)
Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N;
Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poo...
Protein isolation using affinity chromatography
Besselink, T.
Many product or even waste streams in the food industry contain components that may have potential for e.g. functional foods. These streams are typically large in volume and the components of interest are only present at low concentrations. A robust and highly selective separation process should be developed for efficient isolation of the components. Affinity chromatography is such a selective method. Ligands immobilized to a stationary phase (e.g., a resin or membrane) are used to bind the c...
Affinity Purification of Insulin by Peptide-Ligand Affinity Chromatography
Institute of Scientific and Technical Information of China (English)
The affinity heptapeptide (HWWWPAS) for insulin, selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column. The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation. It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography. Nearly 40 mg of insulin could be purified with the 1-mL affinity column. The results revealed the high specificity and capacity of the affinity column for insulin purification. Moreover, based on the analysis of the amino acids in the peptide sequence, shorter peptides were designed and synthesized for insulin chromatography. As a result, HWWPS was found to be a good alternative to HWWWPAS, while the other two peptides with three or four amino acids showed weak affinity for insulin. The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.
Fast and Efficient IMAC Protocol for Phosphopeptide enrichment for phosphoproteomic Studies via LC-MS/MS
McKennan, C.; Spruce, L.; Seeholzer, S; Ding, H.
Recent developments in first dimension High-Performance Liquid Chromatography (HPLC) separation of complex peptide mixtures, followed by a subsequent immobilized metal ion affinity chromatography (IMAC) for phosphopeptide enrichment have shown great promise in both selectivity and quantification of phosphopeptides via LC-MS/MS analysis. The first dimension HPLC, such as hydrophilic interaction chromatography (HILIC) or high pH Reverse Phase chromatography, was employed for its being orthogona...
Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.
Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A
Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801
Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.
Kennedy, Jacob J; Yan, Ping; Zhao, Lei; Ivey, Richard G; Voytovich, Uliana J; Moore, Heather D; Lin, Chenwei; Pogosova-Agadjanyan, Era L; Stirewalt, Derek L; Reding, Kerryn W; Whiteaker, Jeffrey R; Paulovich, Amanda G
A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847
DEFF Research Database (Denmark)
Ye, Juanying; Zhang, Xumin; Young, Clifford; Rudashevskaya, Elena L; Nawrocki, Arek; G. Palmgren, Michael; T. Fuglsang, Anja; Jensen, Ole Nørregaard
progress. Results Fe(III)-IMAC using NTA-silica from Qiagen showed a better performance than two other commercially available resins under the testing conditions. Increase of the acetonitrile content to 60% in loading and washing buffer significantly improved the specificity of IMAC enrichment. It was...... ionization degree References Stensballe, A., Andersen, S. & Jensen, O. N. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis. Proteomics 1, 207-22 (2001)....
Exploring Fluorous Affinity by Liquid Chromatography.
Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto
Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527
Phosphopeptide Purification by IMAC with Fe(III) and Ga(III)
DEFF Research Database (Denmark)
Steen, Hanno; Stensballe, Allan
INTRODUCTIONImmobilized metal ion affinity chromatography (IMAC) makes use of matrix-bound metals to affinity-purify phosphoproteins and phosphopeptides. Commonly used metals in early studies such as Ni(2+), Co(2+), Zn(2+), and Mn(2+) were shown to bind strongly to proteins with a high density of...... histidines. More recently, immobilized Fe(3+), Ga(3+), and Al(3+) metal ions have been used for the selective enrichment of phosphopeptides from complex proteolytic digest mixtures containing both phosphorylated and nonphosphorylated components. The use of a nitrilotriacetic acid (NTA) matrix over...... iminodiacetic-acid-modified matrices has been reported to provide an advantage in selectivity. The development of elution conditions that are directly compatible with MS analysis of the enriched phosphopeptide samples provides the option to interface IMAC and MS online. This protocol describes the enrichment of...
RECOVERY OF CYCLODEXTRIN GLUCANOTRANSFERASE (CGTase USING IMMOBILIZED METAL CHELATING AFFINITY CHROMATOGRAPHY
Directory of Open Access Journals (Sweden)
M. Sivapragasam
Full Text Available Abstract Immobilized metal affinity chromatography (IMAC was chosen as a method of purification for the recovery of CGTase from E. coli homogenate. E. coli harbouring the Bacillus sp. G1 gene expressed extracellularly secreted CGTase into ampicillin supplied LB broth. Culture was pre-purified using SnakeSkin dialysis tubing (3.5 MWCO with an enzyme activity of 147.80 U/mL. Three strategies (A, B and C were employed for the purification of CGTase using column adsorption chromatography with Ni2+-Sepharose resin. Strategy A employed an elution buffer of 50 mM EDTA, pH 7, Strategy B used 0.1 M imidazole, pH 7 and Strategy C employed 45 mM imidazole pH 7 as the elution buffer. Strategy C was found to be most suitable yielding a total CGTase recovery of 87.04% from an initial activity of 147.80 U/mL.
Optimization of conditions for the single step IMAC purification of miraculin from Synsepalum dulcificum.
He, Zuxing; Tan, Joo Shun; Lai, Oi Ming; Ariff, Arbakariya B
In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum. PMID:25794715
Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.
Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej
Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research. PMID:27016113
Hart-Davis, Guy
If you want to get the very most out of your iMac, put this savvy Portable Genius guide to work. Want to make the most of the new Magic Mouse and the latest iLife apps? Set up a wireless network using your iMac's AirPort card? Watch television on your iMac, or show iMac videos and movies on your television? You'll find cool and useful Genius tips, full-color screenshots, and pages of easy-to-access shortcuts and tools that will save you time and let you enjoy your iMac to the max.
Immobilized metal affinity chromatography of phosphorylated proteins using highperformance sorbents
Czech Academy of Sciences Publication Activity Database
Novotná, L.; Hrubý, Martin; Beneš, Milan J.; Kučerová, Z.
Roč. 68, 5-6 (2008), s. 381-386. ISSN 0009-5893 Grant ostatní: GA MŠk(CZ) LC06044 Institutional research plan: CEZ:AV0Z40500505 Keywords : immobilized metal affinity chromatography * phosphorylation * metal ions Subject RIV: CD - Macromolecular Chemistry Impact factor: 1.312, year: 2008
Study of pepsin phosphorylation using immobilized metal affinity chromatography
Czech Academy of Sciences Publication Activity Database
Novotná, L.; Hrubý, Martin; Beneš, Milan J.; Kučerová, Z.
Roč. 31, č. 10 (2008), s. 1662-1668. ISSN 1615-9306 Institutional research plan: CEZ:AV0Z40500505 Keywords : Fe(III) * Ga(III) * immobilized metal affinity chromatography * modification of carboxyl groups Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.746, year: 2008
The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.
Estelrich, J.; Pouplana, R.
Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)
Payne, Matthew John; Holman, Matthew; Alcock, Charles; Osip, David; Schlichting, Hilke
We report the results of our extended campaign to search for occultations of background stars by small (sub-km) Kuiper belt objects (KBOs) using the IMACS instrument on the Magellan Telescope.We implemented a novel shutterless continuous readout mode on the IMACS instrument, with custom-made aperture masks, permitting simultaneous high-speed (~40 Hz) photometry for numerous stars, while minimizing the effects of stellar crowding and sky background. Observing in the southern hemisphere allows us to target the intersection of the ecliptic and galactic planes, where hundreds of stars can be monitored with a single field of view.We observed for a total of ~28 hours spread over eight nights, obtaining over 10,000 star-hours of light curves with per-point SNR > 10. This represents an order of magnitude increase in star hours compared to the previous best ground-based survey by Bianco et al. (2009). Our results allow us to place strong constraints on the surface density of sub-km objects in the Kuiper-Belt, as well as to complement the HST FGS results of Schlichting et al. (2009, 2012).
Chambers, Mark L
Do it all with your iMac and this bestselling For Dummies guide! You're still a little giddy from finally scoring your new iMac, and you can't wait to get started. Even if you're already in love with your iMac, it helps to have a little guidance to really get the most out of this ultimate all-in-one computer. This updated edition of iMac For Dummies is the ideal way to learn the iMac fundamentals from setting up and personalizing your machine to importing files, making FaceTime video calls, surfing the web, using your favorite programs and apps, and everything in between. Trusted Mac guru Mark
Chambers, Mark L
The bestselling guide to the ultimate all-in-one computer—now updated and revised throughout! If you're looking for speed, performance, and power, the iMac is the ultimate all-in-one computer. From its superior performance, powerful operating system, and amazing applications, the iMac is one awesome machine, and the fun, friendly, and approachable style of iMac For Dummies is an ideal way to get started with the basics. You'll learn the fundamentals of the iMac including setting up and customizing your iMac and the software that comes with it, importing files from your old computer, send
Hart-Davis, Guy
The most up-to-date coverage on the latest iMac advice, tools, and shortcuts Cool and useful tips, full-color screenshots, and savvy advice show you how to get the most out of your iMac. Fully updated to cover the iMac's latest features and capabilities, this guide is packed with indispensible information on iLife '09 and Mac OS X Snow Leopard, and shows you how to customize your iMac in a way that it will work best for you.Explores all the bells and whistles of the iMac, including the new Magic Mouse, iLife apps such as iPhoto and iMovie, and Mac OS X Snow LeopardShows yo
Binder, Kate
Cool and useful tips, full-color screenshots, and savvy advice show you how to get the most out of your iMac. You'll discover exactly what the Apple remote is capable of, how to set up your iMac so that it's customized for multiple users, and all there is to know about Mac OS X's built-in organizational tools. This indispensable guide ramps up the pace and offers you easy-to-access shortcuts so you can start enjoying your iMac to the max.
[Fractionation of lymphocytes using affinity chromatography with 9 lectins].
de Dios, I; Manso, M; López-Borrasca, A
Lymphocyte subclasses from normal peripheral blood have been fractionated by affinity chromatography with lectins. Concanavalin A (Con A), Lens culinaris lectin (LC), Pisum sativum lectin (PS), Phaseolus vulgaris lectin (PHA), Dolichos biflours lectin (DB), Glicine max lectin (SBA), Ricinus communis lectin (RCA II), Tetragonolobus purpureus lectin (TP) and Triticum vulgaris lectin (WGA), were coupled to Sepharose 6MB, and lymphocytes labelled with 125I were eluted through the chromatographic columns. The binding of lymphocytes to WGA and SBA lectins was 32% and 13% respectively. The binding to the other lectins tested were found to be between 32% and 13%. When solutions of increasing concentrations of specific sugar were added to the columns a progressive elution of bound lymphocytes was observed. These results indicate the existence of a large range of lymphocyte subclasses, with different binding capacity to lectins, which was a function of the receptor number or/and receptor affinity to each lectin. Furthermore, these two parameters were found to vary in each functional population. Even though all the lymphocytes had lectin receptors, T lymphocytes showed higher affinity for Con A, PHA and TP lectins, while B lymphocytes appeared to be more specific for LC, PS, SBA, DB, RCAII and WGA lectins. PMID:6675094
Affinity chromatography of native and irradiated lactate dehydrogenase
International Nuclear Information System (INIS)
LDH V was irradiated in aqueous solution at a concentration of 1mg/ml in the presence of nitrogen or oxygen. The dose given was 1700 Gy. The irradiated enzyme was separated on 5'AMP Sepharose 4B. The NADH-gradient-eluted fraction was then further studied by determinations of activity, protein, and SH-groups. Gel electrophoresis was also performed on some of the samples. It could be shown that radiation-damaged protein was, overall, less firmly specificially bound. Completely inactivated protein was not bound at all and could be eluted with a buffer immediately after spreading. A wide flat peak was shown in activity determinations of NADH-gradient eluted samples. Radiosensitivity was increased by the presence of oxygen. With the aid of SH-group determination, three further peaks could be identified whereby peaks I and II were bound less strongly, and peak III more strongly than native enzyme. These three peaks are thought to represent different modifications of irradiated LDH. A detailed analysis could be the subject of further investigation. The presence of protein fragments was established by electrophoresis for samples irradiated under oxygen but were not found for samples irradiated under nitrogen. Affinity chromatography is an elegant and safe method for establishing the purity of substances from mixtures. It is, however, only of limited value for studying irradiated proteins. (orig.)
Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.
Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M
Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356
The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique
Landman, A. D.; Landman, N. N.
Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)
Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei
Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis....
Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.
Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W
This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. PMID:24891160
Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.
Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...
Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.
Kudou, Motonori; Okazaki, Fumiyoshi; Asai-Nakashima, Nanami; Ogino, Chiaki; Kondo, Akihiko
Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. PMID:25214227
Optimized IMAC-IMAC protocol for phosphopeptide recovery from complex biological samples
DEFF Research Database (Denmark)
Ye, Juanying; Zhang, Xumin; Young, Clifford; Zhao, Xiaolu; Hao, Qin; Cheng, Lei; Jensen, Ole Nørregaard
that the ionization efficiencies of the phosphoryl group and carboxylic group changed differently when the acetonitrile concentration was increased. A magnified difference was achieved in high acetonitrile content solutions. On the basis of this concept, an optimized phosphopeptide enrichment protocol...... separation of multiply phosphorylated peptides from singly phosphorylated peptides with successive IMAC enrichments. The rational improvements to the IMAC protocol described in this study provide more insights into the factors that affect IMAC performance for phosphopeptide recovery. The improved IMAC......-IMAC method should allow more detailed characterization of phosphoproteins in functional phosphoproteomics research projects....
Adsorption and Step Elution of Urokinase Using, Affinity Chromatography -Comparison of Data with Rate Model Simulation
Institute of Scientific and Technical Information of China (English)
MohammadRezaAboudzadehRovais; JiawenZhu; BinWu
A non-equilibrium chromatographic rate model was employed to simulate the affinity chromatography of urokinase. The chromatography process was developed to a yield of high purity product of urokinase from crude materials. The affinity gel used in the process was prepared by an epichlorohydrin-activation method using epichlorohydrin activated Sepharose 4B as a matrix and p-aminobenzamidine as a ligand. The chromatographic process were numerically simulated and analyzed with the aid of VERSE-LC computer simulator. Considering the basic principles, rate model with the back mixing in column inlet was utilized in simulating and studying the effect of the column inlet pattern on other parameters. Comparison of the simulation results with the experimental data showed that the rate model can be used to describe the affinity chromatography of urokinase in a fixed bed column with satisfactory accuracy.
Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.
Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A
Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625
Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.
Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser
Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166
Comparison of different transition metal ions for immobilized metal affinity chromatography of selenoprotein P from human plasma
DEFF Research Database (Denmark)
Sidenius, U; Farver, O; Jøns, O; Gammelgaard, Bente
Cu2+, Ni2+, Zn2+, Co2+ and Cd2+ were evaluated in metal ion affinity chromatography for enrichment of selenoprotein P, and immobilized Co2+ affinity chromatography was found to be the most selective chromatographic method. The chromatography was performed by fast protein liquid chromatography and...... selenoprotein P. The purity of the protein was determined by SDS-PAGE and by sequencing from polyvinylidene difluoride blots of SDS-PAGE gels....
Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC.
Huang, Sheng-Yu; Tsai, Mei-Ling; Wu, Chin-Jen; Hsu, Jue-Liang; Ho, Shih-Hsin; Chen, Shu-Hui
Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a
A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants
Frank eSainsbury; Philippe V. Jutras; Juan eVorster; Marie-Claire eGoulet; Dominique eMichaud
The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizi...
Displacement affinity chromatography of protein phosphatase one (PP1 complexes
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Gourlay Robert
Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.
Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.
Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong
The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834
Modification of silica-based monolithic capillary columns for boronate affinity chromatography
Czech Academy of Sciences Publication Activity Database
Moravcová, Dana; Planeta, Josef; Kahle, Vladislav; Roth, Michal
2011. P2-G-471-WE. ISBN 978-963-89335-0-8. [International Symposium on High - Performance Liquid Phase Separations and Related Techniques /36./. 19.06.2011-23.06.2011, Budapest] R&D Projects: GA AV ČR IAAX00310701; GA MŠk LC06023 Institutional research plan: CEZ:AV0Z40310501 Keywords : silicagel monolithic column * boronate affinity chromatography Subject RIV: CB - Analytical Chemistry, Separation
Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography.
Kernodle, D S; Zygmunt, D J; McGraw, P A; Chipley, J R
Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-med...
Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome
Energy Technology Data Exchange (ETDEWEB)
Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana
Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.
Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins
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Miriam S. Goyder
Full Text Available We present a top down separation platform for yeast ribosomalproteins using affinity chromatography and capillary electrophoresiswhich is designed to allow deposition of proteins ontoa substrate. FLAG tagged ribosomes were affinity purified, andrRNA acid precipitation was performed on the ribosomes followedby capillary electrophoresis to separate the ribosomalproteins. Over 26 peaks were detected with excellent reproducibility(<0.5% RSD migration time. This is the first reportedseparation of eukaryotic ribosomal proteins using capillaryelectrophoresis. The two stages in this workflow, affinity chromatographyand capillary electrophoresis, share the advantagesthat they are fast, flexible and have small sample requirementsin comparison to more commonly used techniques. This methodis a remarkably quick route from cell to separation that hasthe potential to be coupled to high throughput readout platformsfor studies of the ribosomal proteome. [BMB reports2012; 45(4: 233-238
Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes
Energy Technology Data Exchange (ETDEWEB)
Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. (Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier (France))
The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.
Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes
International Nuclear Information System (INIS)
The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH2)2SS(CH2)2CO-[Ala1, Phe(4N3)8]AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (Kd∼1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors
Sequential elution from IMAC (SIMAC)
DEFF Research Database (Denmark)
Thingholm, Tine E.; Larsen, Martin R.
titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from...... monophosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially...
Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.
Ngo, That T; Narinesingh, Dyer
The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels. PMID:18080884
Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography
International Nuclear Information System (INIS)
Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions
Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.
Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro
A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively. PMID:21919363
In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies
Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette
In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.
Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.
DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U
A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315
Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry
DEFF Research Database (Denmark)
Bahl, Justyna Maria Czarna; Jensen, Søren Skov; Larsen, Martin R;
investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimer's disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of...... phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or...... LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new...
Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.
Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu
An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450
31st IMAC Conference on Structural Dynamics
Adams, Douglas; Carrella, Alex; Mayes, Randy; Rixen, Daniel; Allen, Matt; Cunha, Alvaro; Catbas, Fikret; Pakzad, Shamim; Racic, Vitomir; Pavic, Aleksandar; Reynolds, Paul; Simmermacher, Todd; Cogan, Scott; Moaveni, Babak; Papadimitriou, Costas; Allemang, Randall; Clerck, James; Niezrecki, Christopher; Wicks, Alfred
Topics in Nonlinear Dynamics, Volume 1: Proceedings of the 31st IMAC, A Conference and Exposition on Structural Dynamics, 2013, the first volume of seven from the Conference, brings together contributions to this important area of research and engineering. The collection presents early findings and case studies on fundamental and applied aspects of Structural Dynamics, including papers on: Nonlinear Oscillations Nonlinearities In Practice Nonlinear System Identification: Methods Nonlinear System Identification: Friction & Contact Nonlinear Modal Analysis Nonlinear Modeling & Simulation Nonlinear Vibration Absorbers Constructive Utilization of Nonlinearity.
In vitro-synthesized carbamylated and acetylated hemoglobins interfered in assays of glycohemoglobin by HPLC and electrophoresis but had no effects on results obtained by affinity chromatography and enzyme immunoassay. Correlations between long-term serum urea concentrations and glycohemoglobin perc
Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.
Directory of Open Access Journals (Sweden)
Hua-zhen Wang
Full Text Available Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP could greatly increase the soluble expression level of Glucokinase (GlcK, α-Amylase (Amy and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.
Institute of Scientific and Technical Information of China (English)
YUAN Jing; YAN Husheng; CHENG Xiaohui; HE Binglin
Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, cibacron Blue F3G-A was immobilized,through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N'-Cibacron Blue F3G-A,which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinded poly(vinyl acetate),which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The cibacron Blue F3G-A-immobilized poly(vinyl alcohol)was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.
Advance chromatin extraction improves capture performance of protein A affinity chromatography.
Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete
Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119
Characterization of the galactomannans from Parkinsonia aculeata seeds and their application on affinity chromatography
Directory of Open Access Journals (Sweden)
Ivone Garros-Rosa
Full Text Available Successive aqueous (10 ºC, 25 ºC and 80 ºC and alkaline (1M NaOH; 25 ºC extractions of milled Parkinsonia aculeata endosperms gave rise to four galactomannan fractions. These extractions furnished viscous galactomannans with Man:Gal ratios ranging from 3.1:1; 3.7:1; 4.9:1 and 6.1:1 (P1, P2, P3 and P4, respectively. Fraction P1 was used for structural studies by using methylation analysis, periodate oxidation and 13C-NMR. It showed a linear backbone of beta(1->4 linked D-mannose units, to which single alpha(1->6-linked D-galactose are attached. This galactomannan has Mw 775700 g/mol and intrinsic viscosity of 558 mL/g. The four fractions and the crude endosperm were treated with epichlorydrin and used as matrix for affinity chromatography. All columns tested showed ability to bind lectin samples. The efficiency is related to the degree and pattern of substitution of galactosyl units on the D-mannan backbone.
The Lectin Frontier Database (LfDB, and Data Generation Based on Frontal Affinity Chromatography
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Jun Hirabayashi
Full Text Available Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms—from humans to microorganisms, including viruses—and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's. For Kd determination, an advanced system of frontal affinity chromatography (FAC is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67 of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience.
Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.
Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S
Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924
Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography
Hayakawa, Kou; Katsumata, Noriyuki; Abe, Kiyomi; Hirano, Masahiko; Yoshikawa, Kazuyuki; Ogata, Tsutomu; Horikawa, Reiko; Nagamine, Takeaki
The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs a...
Reaction of Native and Denatured Brucella abortus (S19) Proteins with Antibody Using Affinity Chromatography and Immunoblotting
Karimi, R.; A Mostafaie; B. Tabaraie; Y. Bahrami; J. Abdolalizadeh
Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (m...
Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.
Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland
A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514
Reaction of Native and Denatured Brucella abortus (S19 Proteins with Antibody Using Affinity Chromatography and Immunoblotting
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R. Karimi
Full Text Available Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19 was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting.Upon the results of affinity chromatography and immunoblotting, Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.
Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography.
Hoehenwarter, Wolfgang; Thomas, Martin; Nukarinen, Ella; Egelhofer, Volker; Röhrig, Horst; Weckwerth, Wolfram; Conrath, Uwe; Beckers, Gerold J M
Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)(3)-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO(2)-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. PMID:23172892
Study on peptide-peptide interaction using high-performance affinity chromatography and quartz crystal microbalance biosensor
Institute of Scientific and Technical Information of China (English)
LUO Jia; HUANG YanYan; XIONG ShaoXiang; LIU GuoQuan; ZHAO Rui
The specific interaction between sense and antisense peptides was studied by high-performance affinity chromatography (HPAC) and quartz crystal microbalance (QCM) biosensor. Fragment 1-14 of human interferon-β (hlFN-β) was chosen as sense peptide and its three antisense peptides (AS-IFN 1,AS-IFN 2, and AS-IFN 3) were designed according to the degeneracy of genetic codes. The affinity column was prepared with sense peptide as ligand and the affinity chromatographic behavior was evaluated. Glu-substituted antisense peptide (AS-IFN 3) showed the strongest binding to immobilized sense peptide at pH 7.5. A quartz crystal microbalance-flow injection analysis (QCM-FIA) system was introduced to investigate the recognition process in real-time. The equilibrium dissociation constants between sense peptide and AS-IFN 1, AS-IFN 2 and AS-IFN 3 measured 2.08×10-4, 1.31×10-4 and 2.22×10-5 mol/L, respectively. The mechanism study indicated that the specific recognition between sense peptide and AS-IFN 3 was due to sequence-dependent and multi-modal affinity interaction.
Purification of the Rous sarcoma virus src kinase by casein-agarose and tyrosine-agarose affinity chromatography.
Fukami, Y.; Lipmann, F
A simple and effective purification method for the src kinase, the transforming gene product of Rous sarcoma virus, has been developed by using affinity chromatography on casein-agarose and tyrosine-agarose columns. NaDodSO4/polyacrylamide gel electrophoresis and silver staining analysis showed that the purified kinase preparation was composed of a predominant polypeptide of 60,000-Da. In most of the preparations, however, three minor proteins (54,000, 52,000, and 15,000 Da) were also detecte...
Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.
Hellstern, Simon; Mutzel, Rupert
We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535
Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.
Barbato, O; Sousa, N M; Klisch, K; Clerget, E; Debenedetti, A; Barile, V L; Malfatti, A; Beckers, J F
The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta. PMID:18308351
Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography
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Lina Lu
Full Text Available Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.
Fractionation of phenolase from green table olives (Ascolana tenera var. by immobilized copper affinity chromatography
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Sciancalepore, V.
Full Text Available Phenolase from green table olives (Ascolana tenera var. was fractionated by immobilized copper affinity chromatography. Four chromatographic fractions with high specific activities were obtained. The highest activity was found for the fraction eluted with L-histidine buffer. The four active fractions were characterized by. their response to both specificity and concentration of the substrate.
Fenolasa de aceitunas verdes de mesa de la variedad Ascolana tenera ha sido fraccionada mediante cromatografía de afinidad sobre cobre inmovilizado. Se han obtenido cuatro fracciones cromatográficas con elevada actividad específica. La fracción obtenida con tampon L-histidina fue la que presentó mayor actividad. Las cuatro fracciones activas se caracterizaron por sus respuestas a la especificidad y concentración del sustrato.
β2-Adrenoceptor affinity chromatography and its application in the screening of the active compounds from Semen Armeniacae Amarum
Institute of Scientific and Technical Information of China (English)
ZHENG XiaoHui; ZHAO XinFeng; YANG Rong; WANG ShiXiang; WEI YinMao; ZHENG JianBin
β2-Adrenoceptor (β2-AR) was purified from the rabbit lung tissue by sepharose-salbutamol affinity chromatographic column. To prepare the β2-AR stationary phase, β2-AR was evenly immobilized on the surface of macro-pore silica with a mild chemical coupling method through covalent bond. The reten-tion properties of β2-AR stationary phase were characterized by four ligands, salbutamol sulfate, noradrenaline bitartrate, adrenaline hydrochloride and propranolol hydrochloride, to establish the β2-AR affinity chromatography. Then, the method was used to screen the active compounds from the total extracts of Semen Armeniacae Amarum. The results showed that β2-AR on the surface of the sta-tionary phase could keep its original bioactivity and selectivity. Amygdalin retained in the chroma-tographic column was proved to be the active compound of the total extracts of Semen Armeniacae Amarum. Compared with the existing chromatographic screening approaches, this method showed a good stability and high selectivity. The active compounds which could interact with β2-AR in traditional Chinese medicine (TCM) could be screened efficiently by this method, providing a new way to screen the active compounds in complicated samples such as TCM.
Affinity chromatography of tetanus toxin, tetanus toxoid, and botulinum A toxin on synaptosomes, and differentiation of their acceptors
International Nuclear Information System (INIS)
125I-labelled tetanus toxin and 125I-labelled botulinum A neurotoxin are known to be specifically bound to brain synaptosomes. In order to discriminate between active toxin and inactive admixtures present in the starting material or arising during iodination, synaptosome columns were prepared using bromacetylcellulose and/or kieselgur (Celite) as carriers. Both types of columns adsorb the toxins from low ionic strength medium and release them if the pH and ionic strength are raised. Botulinum toxin was eluted with lower ionic strength than tetanus toxin, and could be freed from nontoxic admixtures. Analysis by affinity chromatography disclosed partially toxoided tetanus toxin in both labelled and unlabelled toxin samples. High concentrations of formaldehyde (0.5%) destroyed both toxicity and affinity to the synaptosomes of tetanus toxin. Low concentrations of formaldehyde (0.05%) yielded a derivative of low toxicity which was still, however less firmly, bound to synaptosomes. Tetanus and botulinum toxin differ by their acceptors. Whereas unlabelled botulinum toxin is unable to compete with labelled tetanus toxin, unlabelled tetanus toxin slightly competes with botulinum toxin. Both labelled toxins display anomalous binding behaviour in that they cannot be displaced completely even with a large excess of unlabelled toxin. (orig.)
Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis
DEFF Research Database (Denmark)
Stensballe, A; Andersen, Søren; Jensen, Ole Nørregaard
determination of phosphorylation sites. The advantages and limitations of the experimental strategy was demonstrated by enrichment, identification and sequencing of phosphopeptides from the model proteins ovalbumin and bovine beta-casein isolated by gel electrophoresis. Furthermore, an autophosphorylation site...... at Ser-3 in recombinant human casein kinase-2 beta subunit was determined. The potential of miniaturized Fe(III)-IMAC and MALDI-MS for characterization of in vivo phosphorylated proteins was demonstrated by identification of tryptic phosphopeptides derived from the human p47/phox phosphoprotein...
Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.
Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S
In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461
Affinely Recursive Functions and Neural Networks
Czech Academy of Sciences Publication Activity Database
Kůrková, Věra; Kainen, P.C.
Atlanta : Georgia Institute of Technology, 1994 - ( Ames , W.), s. 776-779 [IMACS World Congress /14./. Atlanta (US), 11.07.1994-15.07.1994] R&D Projects: GA AV ČR IA23057; GA ČR GA201/93/0427 Keywords : neural networks * affinely recursive functions
Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography
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Claudia Forero
Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.
Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography
Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.
Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.
32nd IMAC Conference and Exposition on Structural Dynamics
Mayes, Randy; Rixen, Daniel; Catbas, Fikret; Atamturktur, H; Moaveni, Babak; Papadimitriou, Costas; Schoenherr, Tyler; Foss, Gary; Niezrecki, Christopher; Allemang, Randall; Kerschen, Gaetan
This critical collection examines a range of topics in modal analysis, from experimental techniques to acoustics to biodynamics, as presented in early findings and case studies from the Proceedings of the 32nd IMAC, A Conference and Exposition on Structural Dynamics, 2014. The collection includes papers in the following general technical research areas: Experimental Techniques, Processing Modal Data, Rotating Machinery, Acoustics, Adaptive Structures, Biodynamics, Damping
Fabrication of Ceramic Membrane Chromatography for Biologics Purification
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Maizirwan Mel
pemfabrikatan membran. Dalam projek ini, serbuk HA dihasilkan menggunakan kanji sebagai agen penghasilan liang. Proses pencirian dilakukan terhadap membran seramik menggunakan radas yang sesuai. Tiga parameter proses pemfabrikatan (peratusan berat kanji, tekanan padatan dan suhu pensinteran dimanipulasikan untuk mendapatkan prestasi membran yang optima. Membran yang difabrikatkan diletakkan dalam sistem FPLC (Fast Protein Liquid Chromatography untuk diuji prestasinya sebagai membran serap. Proses IMAC (Immobilized Metal Affinity Chromatography dijalankan dengan memegunkan ion Ni2+ pada permukaan zarah membran. Nucleoprotein dari NDV (Newcastle disease virus digunakan untuk menguji kebolehan membran terikat dengan protein yang dilabelkan dengan Hisditina. Set parameter proses yang optima yang menghasilkan keliangan tertinggi dan kromatogram yang baik ditentukan pada berat kanji 5 %, tekanan padatan 3000 psi dan suhu pensinteran 1100°C.KEYWORDS: Membrane Chromatography, Porous Ceramic Membrane, IMAC, Hydroxyapatite, Chromatography.
TiO2-ZrO2 affinity chromatography polymeric microchip for phosphopeptide enrichment and separation.
Tsougeni, Katerina; Zerefos, Panagiotis; Tserepi, Angeliki; Vlahou, Antonia; Garbis, Spiros D; Gogolides, Evangelos
We fabricated a TiO(2)-ZrO(2) affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono- and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of β-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO(2)-ZrO(2) stationary phase was characterized by X-ray diffraction and was found to be mostly TiO(2) and ZrO(2) in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of >1.4 μg (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 ± 3%, and can be used with small samples (less than 0.1 μL depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 μg and sample volumes of 1-10 μL), and to on-chip efforts based on packed bed and frit formats. PMID:21796280
Metal oxide affinity chromatography platform-polydopamine coupled functional two-dimensional titania graphene nanohybrid for phosphoproteome research.
Yan, Yinghua; Sun, Xueni; Deng, Chunhui; Li, Yan; Zhang, Xiangmin
In this work, a facile route was initially developed for preparation of a novel metal oxide affinity chromatography (MOAC) material by grafting titania nanoparticles on polydopamine (PD)-coated graphene (denoted as G@PD@TiO2). In the first step, self-assemble polymerization of dopamine on graphene was performed in basic solution at room temperature, which not only offered the coupling linker between titania and graphene but also improved the hydrophilicity and biological compatibility of the nanohybrids. Thereafter, the titania nanoparticles were grafted on the surface of the PD-coated graphene via a simple hydrothermal treatment. The as-prepared G@PD@TiO2 nanohybrids exhibited high sensitivity (detection limit of 5 fmol) and high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/nonphosphopeptides (1:1000). Moreover, the as-prepared nanohybrids were also investigated for enrichment of phosphopeptides from real biological samples (human serum and mouse brain). A total number of 556 phosphorylation sites were identified from the digest of mouse brain proteins, showing great potential in the practical application. PMID:24673251
Use of Aleuria alantia Lectin Affinity Chromatography to Enrich Candidate Biomarkers from the Urine of Patients with Bladder Cancer
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Sarah R. Ambrose
Full Text Available Developing a urine test to detect bladder tumours with high sensitivity and specificity is a key goal in bladder cancer research. We hypothesised that bladder cancer-specific glycoproteins might fulfill this role. Lectin-ELISAs were used to study the binding of 25 lectins to 10 bladder cell lines and serum and urine from bladder cancer patients and non-cancer controls. Selected lectins were then used to enrich glycoproteins from the urine of bladder cancer patients and control subjects for analysis by shotgun proteomics. None of the lectins showed a strong preference for bladder cancer cell lines over normal urothlelial cell lines or for urinary glycans from bladder cancer patients over those from non-cancer controls. However, several lectins showed a strong preference for bladder cell line glycans over serum glycans and are potentially useful for enriching glycoproteins originating from the urothelium in urine. Aleuria alantia lectin affinity chromatography and shotgun proteomics identified mucin-1 and golgi apparatus protein 1 as proteins warranting further investigation as urinary biomarkers for low-grade bladder cancer. Glycosylation changes in bladder cancer are not reliably detected by measuring lectin binding to unfractionated proteomes, but it is possible that more specific reagents and/or a focus on individual proteins may produce clinically useful biomarkers.
Institute of Scientific and Technical Information of China (English)
YUANJing; YANHusheng; 等
Immobilized triazine dye affinity chromatography has been widely used for protein purification.In this paper,Cibacron Blue F3G-4 was immobilized,through a spacer arm,onto a rigid hydroplhilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol)with 6-aminohexyl-N'-Cibacron Blue F3G-A,which was obtained by reacting Cibacron Blue F3G-4 with excess of 1,6-diaminohexane,in a pH 8.6buffer,The epoxy-group-containing poly(vinyl alcohol)was prepared by treating macroporous poly(vinyl alcohol)with excess epichlorohydrin in the presence of NaOH in dimethyl sulfoxide.The macroporous poly(vinyl alcohol)was prepared by hydrolysis of macroporous crosslinked poly(vinyl acetate),which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents.The Cibacron Blue F3G-A-immobilized poly(vinyl alcohol)was packed in a stainless steel column (250×5mm I.D.)and the chromatographic behaviors of several proteins(cytochrome c, lysozyme,bovine serum albumin,insulin,and lactate dehydrogenase)were determined.
Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques
Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.
Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.
Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography
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Gleyce Alves Machado
Full Text Available The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene ethanol Triton X-114 (TX-114 partitioning in the diagnosis of human neurocysticercosis (NCC. Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound and aqueous (AJ unbound fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.
Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography.
Anderson, D J; Branum, E L; O'Brien, J F
To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively). PMID:2302767
Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites
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Weckwerth Wolfram
Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to
Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K S; Hage, David S.
This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclaz...
Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography
DEFF Research Database (Denmark)
Prentø, Jannick Cornelius; Bukh, Jens
Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However,...... physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development....
34th IMAC, A Conference and Exposition on Structural Dynamics
Brandt, Anders; Singhal, Raj
Nonlinear Dynamics, Volume 1. Proceedings of the 34th IMAC, A Conference and Exposition on Dynamics of Multiphysical Systems: From Active Materials to Vibroacoustics, 2016, the fi rst volume of ten from the Conference, brings together contributions to this important area of research and engineering. Th e collection presents early fi ndings and case studies on fundamental and applied aspects of Structural Dynamics, including papers on: • Nonlinear Oscillations • Nonlinear Modal Analysis • Nonlinear System Identifi cation • Nonlinear Modeling & Simulation • Nonlinearity in Practice • Nonlinearity in Multi-Physics Systems • Nonlinear Modes and Modal Interactions.
30th IMAC, A Conference on Structural Dynamics
Catbas, FN; Mayes, R; Rixen, D; Griffith, DT; Allemang, R; Clerck, J; Klerk, D; Simmermacher, T; Cogan, S; Chauhan, S; Cunha, A; Racic, V; Reynolds, P; Salyards, K; Adams, D; Kerschen, G; Carrella, A; Voormeeren, SN; Allen, MS; Horta, LG; Barthorpe, R; Niezrecki, C; Blough, JR; Vol.1 Topics on the Dynamics of Civil Structures; Vol.2 Topics in Experimental Dynamics Substructuring and Wind Turbine Dynamics; Vol.3 Topics in Nonlinear Dynamics; Vol.4 Topics in Model Validation and Uncertainty Quantification; Vol.5 Topics in Modal Analysis I; Vol.6 Topics in Modal Analysis II
Topics on the Dynamics of Civil Structures, Volume 1, Proceedings of the 30th IMAC, A Conference and Exposition on Structural Dynamics, 2012, the first volume of six from the Conference, brings together 45 contributions to this important area of research and engineering. The collection presents early findings and case studies on fundamental and applied aspects of Structural Dynamics, including papers on: Human Induced Vibrations Bridge Dynamics Operational Modal Analysis Experimental Techniques and Modeling for Civil Structures System Identification for Civil Structures Method and Technologies for Bridge Monitoring Damage Detection for Civil Structures Structural Modeling Vibration Control Method and Approaches for Civil Structures Modal Testing of Civil Structures.
Lanthanide-IMAC enrichment of carbohydrates and polyols.
Schemeth, Dieter; Rainer, Matthias; Messner, Christoph B; Rode, Bernd M; Bonn, Günther K
In this study a new type of immobilized metal ion affinity chromatography resin for the enrichment of carbohydrates and polyols was synthesized by radical polymerization reaction of vinyl phosphonic acid and 1,4-butandiole dimethacrylate using azo-bis-isobutyronitrile as radical initiator. Interaction between the chelated trivalent lanthanide ions and negatively charged hydroxyl groups of carbohydrates and polyols was observed by applying high pH values. The new method was evaluated by single standard solutions, mixtures of standards, honey and a more complex extract of Cynara scolymus. The washing step was accomplished by acetonitrile in excess volumes. Elution of enriched carbohydrates was successfully performed with deionized water. The subsequent analysis was carried out with matrix-free laser desorption/ionization-time of flight mass spectrometry involving a TiO2 -coated steel target, especially suitable for the measurement of low-molecular-weight substances. Quantitative analysis of the sugar alcohol xylitol as well as the determination of the maximal loading capacity was performed by gas chromatography in conjunction with mass spectrometric detection after chemical derivatization. In a parallel approach quantum mechanical geometry optimizations were performed in order to compare the coordination behavior of various trivalent lanthanide ions. PMID:24097333
Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.
Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V
The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions. PMID:17223789
Isolation of specific anti-T3 and anti-T4 antibodies by affinity chromatography and their application for solid phase radioimmunoassay
International Nuclear Information System (INIS)
For laboratory, clinical and experimental purposes highly purified and high avidity antibodies, devoid of other plasma proteins, are required. Hitherto physical and chemical methods have not provided antibodies of sufficient purity, and it was only the introduction of affinity chromatography which achieved antibodies of required purity. Various suports /Affi-Gel 10, CNBr-activated Sepharose 4B, AH-Sepharose 4B and Epoxy-activated Sepharose 6B/ were tried for immobilization of iodothyronines. From these materials Epoxy-activated Sepharose 6B gave most satisfactory results, hence some details of the coupling procedure are given
Affinity Chromatography Method for Determination of Binding of Drugs to Melanin and Evaluation of Side Effect Potential of Antipsychotic Agents
Marszałł, Michał Piotr; Proszowska, Anna; Buciński, Adam; Kaliszan, Roman
The extrapyramidal side effect parameters of typical and atypical antypsychotics were correlated with affinity chromatographic data determined on the melanin-based column. The chromatographic study was performed according to the hypothesis that extrapyramidal symptoms (EPS) as side effects of the use of antipsychotic drugs at clinically effective doses are correlated to the affinity of these drugs to neuromelanin. For that aim the polymerization product of L-DOPA (melanin) was immobilized ont...
Isolation and Identification of Zn2+ Binding Proteins in Streptococcus pyogenes by IMAC and LC-MS/MS%利用IMAC与LC-MS/MS技术分离和鉴定化脓链球菌中的锌结合蛋白
Institute of Scientific and Technical Information of China (English)
阳小燕; 银兴峰; 张留辉; 贺翔; 孙雪松
Streptococcus pyogenes is a gram-positive pathogen that causes a variety of human diseases. The zinc binding proteins of S. pyogenes were enriched through Immobilized-metal affinity chromatography (IMAC) .The proteins were further digested in-solution and analyzed by the advanced LC-MS/MS. In total, forty-eight zinc binding proteins involved in protein translation, glucose metabolism, metal transport, oxidation and other physiological processes were identified. The data presented here are helpful in understanding of the pathological mechanisms of S. pyogenes and facilitate the discovery of potential antibacterial drug targets and vaccine candidates.%化脓链球菌是一种革兰氏阳性致病菌,能引起一系列疾病.运用固定化金属离子亲和层析技术(IMAC)富集化脓链球菌中锌结合蛋白,进行溶液酶解,利用先进的LC-MS/MS技术对富集的锌结合蛋白进行质谱分析.鉴定到48个在化脓链球菌中与锌结合相关蛋白,这些蛋白主要涉及到蛋白翻译、糖代谢、金属转运、氧化等重要生理过程.这些结果对于深入研究化脓链球菌的致病机制,寻找新的抗菌药物作用靶点和疫苗候选物具有重要意义.
High-performance liquid chromatography of amino acids, peptides and proteins. CIII. Mass transfer resistances in ion-exchange and dye-affinity chromatography of proteins.
Johnston, A; Hearn, M T
Adsorption equilibria and rate kinetics have been investigated for the binding of several proteins, with different molecular geometries, to several ion-exchange and dye-affinity chromatographic resins with varying pore size and protein accessibilities. The pore geometry was shown to play a significant role in the protein capacity and loadability of both the ion-exchange and dye-affinity resins. For example the Fractogel HW75-Cibacron Blue F3GA affinity sorbent had the greatest capacity for the small protein, lysozyme, compared to the other Fractogel HW-Cibacron Blue F3GA sorbents, and similarly, the ion-exchange resins, such as DEAE-Fractogel 65, bound more human serum albumin (HSA), as opposed to the larger protein, ferritin. The apparent diffusion of protein from the bulk phase to the ligands/ionic sites was calculated to be considerably restricted when the pore to protein size ratio was small, as is the case of DEAE Fractogel 65/ferritin system, and the dye-affinity Fractogel HW55/HSA system. In these circumstances, pore diffusivity was calculated to be up to 100-fold smaller than bulk diffusivity. PMID:2229222
Hydrophilic Nb{sup 5+}-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides
Energy Technology Data Exchange (ETDEWEB)
Sun, Xueni; Liu, Xiaodan; Feng, Jianan [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Li, Yan, E-mail: firstname.lastname@example.com [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Deng, Chunhui [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Duan, Gengli [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China)
Highlights: • A new IMAC material (Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) was synthesized. • The strong magnetic behaviors of the microspheres ensure fast and easy separation. • The enrichment ability was tested by human serum and nonfat milk. • The results were compared with other IMAC materials including the commercial kits. • All results proved the good enrichment ability, especially for multiphosphopeptides. - Abstract: Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe{sub 3}O{sub 4}@polydopamine-Nb{sup 5+} (denoted as Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe{sub 3}O{sub 4}@PD-Ti{sup 4+} microspheres, the Fe{sub 3}O{sub 4}@PD-Nb{sup 5+} microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.
Enrichment and separation of mono- and multiply phosphorylated peptides using sequential elution from IMAC prior to mass spectrometric analysis
DEFF Research Database (Denmark)
Thingholm, Tine E; Jensen, Ole N; Larsen, Martin R
Phospho-proteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., immobilized metal affinity chromatography and titanium dioxide...
The combination of lectin affinity chromatography, gel electrophoresis and mass spectrometry in the study of plant glycoproteome: Preliminary insights
Czech Academy of Sciences Publication Activity Database
Laštovičková, Markéta; Smětalová, D.; Bobálová, Janette
Roč. 73, Suppl 1 (2011), S113–S122. ISSN 0009-5893 R&D Projects: GA MŠk 1M0570; GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : lectin chromatography * MALDI-TOF mass spectrometry * Glycoproteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.195, year: 2011
Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.
Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U
Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO. PMID:26851523
An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography
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Büssow Konrad
Full Text Available Abstract Background Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. Results We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag or glutathione S-transferase (GST-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. Conclusion A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies. His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.
Hydrophilic Nb⁵⁺-immobilized magnetic core-shell microsphere--A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides.
Sun, Xueni; Liu, Xiaodan; Feng, Jianan; Li, Yan; Deng, Chunhui; Duan, Gengli
Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe3O4@polydopamine-Nb(5+) (denoted as Fe3O4@PD-Nb(5+)) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA=1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe3O4@PD-Ti(4+) microspheres, the Fe3O4@PD-Nb(5+) microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials. PMID:26092339
Designed synthesis of Graphene @titania @mesoporous silica hybrid material as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides.
Yao, Jizong; Sun, Nianrong; Deng, Chunhui; Zhang, Xiangming
In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5amol/μL (a total amount of 1fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully. PMID:26838411
Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography
Energy Technology Data Exchange (ETDEWEB)
Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.
Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.
Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.
Li, Qian; Zhang, Tianlong; Bian, Liujiao
Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties. PMID:26882128
Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.
Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A
Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG. PMID:26476866
Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.
Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen
The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug. PMID:26851087
Reproducible Automated Phosphopeptide Enrichment Using Magnetic TiO2 and Ti-IMAC
Tape, Christopher J.; Worboys, Jonathan D.; Sinclair, John; Gourlay, Robert; Vogt, Janis; McMahon, Kelly M.; Trost, Matthias; Lauffenburger, Douglas A.; Lamont, Douglas J.; Jørgensen, Claus
Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demo...
Sequential Elution from IMAC (SIMAC): An Efficient Method for Enrichment and Separation of Mono- and Multi-phosphorylated Peptides.
Thingholm, Tine E; Larsen, Martin R
Phosphoproteomics relies on methods for efficient purification and sequencing of phosphopeptides from highly complex biological systems, especially when using low amounts of starting material. Current methods for phosphopeptide enrichment, e.g., Immobilized Metal ion Affinity Chromatography and titanium dioxide chromatography provide varying degrees of selectivity and specificity for phosphopeptide enrichment. The number of multi-phosphorylated peptides identified in most published studies is rather low. Here we describe a protocol for a strategy that separates mono-phosphorylated peptides from multiply phosphorylated peptides using Sequential elution from Immobilized Metal ion Affinity Chromatography. The method relies on the initial enrichment and separation of mono- and multi-phosphorylated peptides using Immobilized Metal ion Affinity Chromatography and a subsequent enrichment of the mono-phosphorylated peptides using titanium dioxide chromatography. The two separate phosphopeptide fractions are then subsequently analyzed by mass spectrometric methods optimized for mono-phosphorylated and multi-phosphorylated peptides, respectively, resulting in improved identification of especially multi-phosphorylated peptides from a minimum amount of starting material. PMID:26584924
Optimization of Traffic System using TCL Algorithm through FMSA and IMAC Agents
Directory of Open Access Journals (Sweden)
T.Karthikeyan , S.Sujatha
Full Text Available Based on Agent Technology, Service Oriented Architecture and RFID,this paper puts forward a 'real time paradigm' traffic congestion control strategy in metropolitan cities. This strategy uses TCL algorithm in order to provide optimal traffic performance evaluation considering the parameter, velocity of vehicles by implementing RMI & SOAP interfaces. This building block of traffic and transportation system is more suited for the proposed mobile agents FMSA(Fixed Monitor Stationary Agent & IMAC(Interactive Mobile Agent for Client because this model works on the Agent technology that can be developed using Java and JADE Agent platform under dynamic changing environments. The developed FMSA & IMAC agents makes real time decisions for choosing the best path in road traffic network to avoid congestion by considering the parameters for optimization like mainly the velocity of vehicles in the existing lane. This paper also compares the experimental results obtained by the system before and after the implementation of FMSA & IMAC agents to prove the significant performance for finding the best path by confirming the better efficiency & scalability improvement of our framework.
Validation of Process for Virus Removal from Recombinant Monoclonal Antibody Product by Affinity Chromatography%重组单抗药物亲和层析病毒去除工艺验证
Institute of Scientific and Technical Information of China (English)
程立均; 马翠卿; 杨建岭; 魏敬双; 张世雄; 刘俊乐; 魏林; 贾茜
Objective To verify the process for virus removal from recombinant monoclonal antibody product by affinity chromatography.Methods The efficacies of virus removal from recombinant monoclonal antibody against rabies virus by novel and traditional rProtein A Sepharose Fast Flow (rProtein A SFF) chromatography were evaluated using influenza virus subtype H1N1, herpes simplex virus type 1 and adenovirus type 5 as model viruses. Results All the titers of three kinds of model viruses decreased by more than 4. 0 log10 after purification by novel and traditional rProtein A SFF chromatography. Conclusion The rProtein A SFF affinity chromatography is effective in removal of potential virus contamination from recombinant monoclonal antibody products.%目的 对重组单抗药物亲和层析病毒去除工艺进行验证.方法 以甲型流感病毒H1N1亚型、单纯疱疹病毒1型和腺病毒5型作为指示病毒,分别使用新、旧rProtein A Sepharose Fast Flow(rProtein A SFF)层析介质考察重组抗狂犬病病毒单抗亲和层析步骤对病毒的去除效果.结果 经新、旧rProtein A SFF介质亲和层析后,3种指示病毒的滴度均下降4.0log10以上.结论 rProtein A SFF亲和层析工艺能有效去除重组单抗药物的潜在病毒污染.
Cockpit Interfaces, Displays, and Alerting Messages for the Interval Management Alternative Clearances (IMAC) Experiment
Baxley, Brian T.; Palmer, Michael T.; Swieringa, Kurt A.
This document describes the IM cockpit interfaces, displays, and alerting capabilities that were developed for and used in the IMAC experiment, which was conducted at NASA Langley in the summer of 2015. Specifically, this document includes: (1) screen layouts for each page of the interface; (2) step-by-step instructions for data entry, data verification and input error correction; (3) algorithm state messages and error condition alerting messages; (4) aircraft speed guidance and deviation indications; and (5) graphical display of the spatial relationships between the Ownship aircraft and the Target aircraft. The controller displays for IM will be described in a separate document.
Isolation of pregnancy-associated glycoproteins (PAG) from water buffalo (Bubalus bubalis) placenta by use of Vicia villosa bound agarose affinity chromatography
Beckers, J. F.; Malfatti, A.; V. Barile; Debenedetti, A.; Clerget, E.; K. Klisch; Sousa, N.M.; O. Barbato
The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalis bubalis). After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG) were enriched by Vicia villosa agarose (VVA) affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8...
International Nuclear Information System (INIS)
The IMACS Cluster Building Survey uses the wide field spectroscopic capabilities of the IMACS spectrograph on the 6.5 m Baade Telescope to survey the large-scale environment surrounding rich intermediate-redshift clusters of galaxies. The goal is to understand the processes which may be transforming star-forming field galaxies into quiescent cluster members as groups and individual galaxies fall into the cluster from the surrounding supercluster. This first paper describes the survey: the data taking and reduction methods. We provide new calibrations of star formation rates (SFRs) derived from optical and infrared spectroscopy and photometry. We demonstrate that there is a tight relation between the observed SFR per unit B luminosity, and the ratio of the extinctions of the stellar continuum and the optical emission lines. With this, we can obtain accurate extinction-corrected colors of galaxies. Using these colors as well as other spectral measures, we determine new criteria for the existence of ongoing and recent starbursts in galaxies.
The IMACS Cluster Building Survey. I. Description of the Survey and Analysis Methods
Oemler Jr., Augustus; Dressler, Alan; Gladders, Michael G.; Rigby, Jane R.; Bai, Lei; Kelson, Daniel; Villanueva, Edward; Fritz, Jacopo; Rieke, George; Poggianti, Bianca M.; Vulcani, Benedetta
The IMACS Cluster Building Survey uses the wide field spectroscopic capabilities of the IMACS spectrograph on the 6.5 m Baade Telescope to survey the large-scale environment surrounding rich intermediate-redshift clusters of galaxies. The goal is to understand the processes which may be transforming star-forming field galaxies into quiescent cluster members as groups and individual galaxies fall into the cluster from the surrounding supercluster. This first paper describes the survey: the data taking and reduction methods. We provide new calibrations of star formation rates (SFRs) derived from optical and infrared spectroscopy and photometry. We demonstrate that there is a tight relation between the observed SFR per unit B luminosity, and the ratio of the extinctions of the stellar continuum and the optical emission lines.With this, we can obtain accurate extinction-corrected colors of galaxies. Using these colors as well as other spectral measures, we determine new criteria for the existence of ongoing and recent starbursts in galaxies.
Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.
Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves
This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution. PMID:26974869
High-productivity membrane adsorbers: Polymer surface-modification studies for ion-exchange and affinity bioseparations
Chenette, Heather C. S.
membrane adsorbers were found to have a static binding capacity for con A (6.0 mg/mL) that is nearly the same as the typical dextran-based separation media used in practice. Binding under dynamic conditions was tested using flow rates of 0.1-1.0 mL/min. No bound lectin was observed for the higher flow rate. The first Damkohler number was used to assess whether adsorption kinetics or mass transport contributed the limitation to conA binding. Analyses indicate that this system is not limited by the accessibility of the binding sites, but by the inherently low rate of adsorption of conA onto the glycopolymer. The research described in Chapter 4 focuses on reaction chemistry experiments to incorporate a phosphonate-based polymer in the membrane platform to develop a new class of affinity adsorbers that function based on their affinity for Arginine (Arg) amino acid residues. The hypothesis was that benzyl phosphonate-containing functional polymers would form strong complexes with Arg-rich proteins as a result of multivalent binding. Introducing a new class of affinity membranes for purification of Arg-rich and Arg-tagged proteins may have an impact similar to the introduction of immobilized metal ion affinity chromatography (IMAC), which would be a significant achievement. Using Arg-tags would overcome some of the associated drawbacks of using metal ions in IMAC. Additionally, some cell penetrating peptides are said to be Arg-rich, and this would be a convenient feature to exploit for their isolation and purification. Lysozyme was used as a model Arg-rich protein. The affinity membranes show a static binding capacity of 3 mg/mL. (Abstract shortened by UMI.)
The IMACS Cluster Building Survey: V. Further Evidence for Starburst Recycling from Quantitative Galaxy Morphologies
Abramson, Louis E; Gladders, Michael D; Oemler,, Augustus; Poggianti, Bianca M; Monson, Andrew; Persson, Eric; Vulcani, Benedetta
Using J and K_s band imaging obtained as part of the IMACS Cluster Building Survey (ICBS) we measure S\\'ersic indices for 2160 field and cluster galaxies at 0.31 < z < 0.54. We compare the distributions for spectroscopically determined passive, continuously starforming, starburst, and poststarburst systems and show that previously established spatial/statistical connections between these types extend to their gross morphologies. Outside of cluster cores, we find close structural ties between starburst and continuously starforming, as well as poststarburst and passive types, but not between starbursts and poststarbursts. These results independently support two conclusions presented in a previous ICBS paper (Dressler et al.): 1) most starbursts are the product of a non-disruptive triggering mechanism that is insensitive to global environment, such as minor-mergers; 2) starbursts and poststarbursts largely represent transient phases in the lives of "normal" starforming and quiescent galaxies, respectively,...
Isolation of pregnancy-associated glycoproteins (PAG from water buffalo (Bubalus bubalis placenta by use of Vicia villosa bound agarose affinity chromatography
Directory of Open Access Journals (Sweden)
J.F. Beckers
Full Text Available The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalis bubalis. After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG were enriched by Vicia villosa agarose (VVA affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8 kDa and from 57.8 to 80.9 kDa in the mid- and late- pregnancy placenta respectively. Aminoterminal microsequencing of proteins allowed the identification of three distinct wbPAG sequences wich have ben deposed in the SwissProt database: RGSXLTIHPLRNIRDFFYUG (Acc. n. P85048, RGSXLTILPLRNIID (P85049 and RGSXLTHLPLRNI (P85050. Their comparison to those previously identified revealed that two of them were new since they have not been described yet. Our results confirm the suitability of VVA chromatography in enrichment of multiple PAG molecules expressed in buffalo placenta. Productions of specific antisera can be very useful in immonoistochemical and immunocyitochemical studies of PAG expression in fetomaternal interfaces. Purified native PAG are also required for development on specific immoassays (RIA/ELISA currently used for pregnancy diagnosis and physiological investigation in farm animal.
Short communication: Identification of iron-binding peptides from whey protein hydrolysates using iron (III)-immobilized metal ion affinity chromatography and reversed phase-HPLC-tandem mass spectrometry.
Cruz-Huerta, Elvia; Martínez Maqueda, Daniel; de la Hoz, Lucia; da Silva, Vera S Nunes; Pacheco, Maria Teresa Bertoldo; Amigo, Lourdes; Recio, Isidra
Peptides with iron-binding capacity obtained by hydrolysis of whey protein with Alcalase (Novozymes, Araucaria, PR, Brazil), pancreatin, and Flavourzyme (Novozymes) were identified. Hydrolysates were subjected to iron (III)-immobilized metal ion affinity chromatography, and the bound peptides were sequenced by mass spectrometry. Regardless of the enzyme used, the domains f(42-59) and f(125-137) from β-lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case of peptides derived from α-lactalbumin, with sequences deriving from diverse regions. Iron-bound peptides exhibited common structural characteristics, such as an abundance of Asp, Glu, and Pro, as revealed by mass spectrometry and AA analysis. In conclusion, this characterization of iron-binding peptides helps clarify the relationship between peptide structure and iron-chelating activity and supports the promising role of whey protein hydrolysates as functional ingredients in iron supplementation treatments. PMID:26601589
On-line coupling of surface plasmon resonance optical sensing to size-exclusion chromatography for affinity assessment of antibody samples.
Lakayan, Dina; Haselberg, Rob; Niessen, Wilfried M A; Somsen, Govert W; Kool, Jeroen
Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. PMID:27215465
Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection.
Takeda, N; Gotoh, M; Matsuoka, T
An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 µg kg(-1) (DAN) to 6.5 µg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 µg kg(-1)) and shrimp (ENR 20 µg kg(-1)). PMID:21749230
International Nuclear Information System (INIS)
Using J- and Ks-band imaging obtained as part of the IMACS Cluster Building Survey (ICBS), we measure Sérsic indices for 2160 field and cluster galaxies at 0.31 < z < 0.54. Using both mass- and magnitude-limited samples, we compare the distributions for spectroscopically determined passive, continuously star-forming, starburst, and post-starburst systems and show that previously established spatial and statistical connections between these types extend to their gross morphologies. Outside of cluster cores, we find close structural ties between starburst and continuously star-forming, as well as post-starburst and passive types, but not between starbursts and post-starbursts. These results independently support two conclusions presented in Paper II of this series: (1) most starbursts are the product of a non-disruptive triggering mechanism that is insensitive to global environment, such as minor mergers; (2) starbursts and post-starbursts generally represent transient phases in the lives of 'normal' star-forming and quiescent galaxies, respectively, originating from and returning to these systems in closed 'recycling' loops. In this picture, spectroscopically identified post-starbursts constitute a minority of all recently terminated starbursts, largely ruling out the typical starburst as a quenching event in all but the densest environments
Flight Crew Responses to the Interval Management Alternative Clearances (IMAC) Experiment
Baxley, Brian T.; Wilson, Sara R.; Swieringa, Kurt A.; Roper, Roy D.
Interval Management Alternative Clearances (IMAC) was a human-in-the-loop simulation experiment conducted to explore the efficacy and acceptability of three IM operations: CAPTURE, CROSS, and MAINTAIN. Two weeks of data collection were conducted, with each week using twelve subject pilots and four subject controllers flying ten high-density arrival scenarios into the Denver International Airport. Overall, both the IM operations and procedures were rated very favorably by the flight crew in terms of acceptability, workload, and pilot head down time. However, several critical issues were identified requiring resolution prior to real-world implementation, including the high frequency of IM speed commands, IM speed commands requiring changes to aircraft configuration, and ambiguous IM cockpit displays that did not trigger the intended pilot reaction. The results from this experiment will be used to prepare for a flight test in 2017, and to support the development of an advanced IM concept of operations by the FAA (Federal Aviation Agency) and aviation industry.
Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.
Sriwanthana, B; Island, M D; Maneval, D; Mobley, H L
Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations. PMID:7961442
DEFF Research Database (Denmark)
Guglielmi, Michel; Johannesen, Hl
, Essex, Hertfordshire, Norfolk and Suffolk. Research found that there was a lack of identity or sense of belonging and nothing anchoring people to the region as a whole. Common affinity is somehow forced to the people of East England and thereby we came to the conclusion that a single landmark or a...... a sense of belonging to people sharing deterritorialized synchronic experiences. But at the same time, the immersion experience is highly low tech and desperately analog, mainly based on fabulation, cartoons, and mushrooms growing in local forests. It ultimately appeals to the experienced sense of...
Optimization of MALDI-TOF MS Detection for Enhanced Sensitivity of Affinity-Captured Proteins Spanning a 100 kDa Mass Range
Gatlin-Bunai, Christine L.; Lisa H. Cazares; Cooke, William E.; Semmes, Oliver J.; Malyarenko, Dariya I.
Analysis of complex biological samples by MALDI-TOF mass spectrometry has been generally limited to the detection of low-mass protein (or protein fragment) peaks. We have extended the mass range of MALDI-TOF high-sensitivity detection by an order of magnitude through the combined optimization of instrument parameters, data processing, and sample preparation procedures for affinity capture. WCX, C3, and IMAC magnetic beads were determined to be complementary and most favorable for broad mass r...
Affinity purification of aprotinin from bovine lung.
Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun
An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462
The purification of affinity-labelled active-site peptides
International Nuclear Information System (INIS)
The isolation of the labelled peptide from the protein digest, following the affinity labelling of the active sites of enzymes or antibodies, is described. Single-step affinity chromatography utilises the affinity of the native enzymes or antibody for the ligand used to label the same protein. The labelled peptide is the only one in the digest that displays affinity for the immobilised protein and can be released with eluants that dissociate the protein-ligand complex. (Auth.)
Limitations in SELDI-TOF MS whole serum proteomic profiling with IMAC surface to specifically detect colorectal cancer
International Nuclear Information System (INIS)
Surface enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis on serum samples was reported to be able to detect colorectal cancer (CRC) from normal or control patients. We carried out a validation study of a SELDI-TOF MS approach with IMAC surface sample processing to identify CRC. A retrospective cohort of 338 serum samples including 154 CRCs, 67 control cancers and 117 non-cancerous conditions was profiled using SELDI-TOF-MS. No CRC 'specific' classifier was found. However, a classifier consisting of two protein peaks separates cancer from non-cancerous conditions with high accuracy. In this study, the SELDI-TOF-MS-based protein expression profiling approach did not perform to identify CRC. However, this technique is promising in distinguishing patients with cancer from a non-cancerous population; it may be useful for monitoring recurrence of CRC after treatment
J. Navarro; Sancho, C.; Sancho, P.
A functor of sets $\\mathbb X$ over the category of $K$-commutative algebras is said to be an affine functor if its functor of functions, $\\mathbb A_{\\mathbb X}$, is reflexive and $\\mathbb X=\\Spec \\mathbb A_{\\mathbb X}$. We prove that affine functors are equal to a direct limit of affine schemes and that affine schemes, formal schemes, the completion of affine schemes along a closed subscheme, etc., are affine functors. Endowing an affine functor $\\mathbb X$ with a functor of monoids structure...
亲和层析微柱法测定肝癌特异性AFP及其在肝癌诊断中的临床价值%Quantitative analysis of hepatoma-specific α-fetoprotein (HS-AFP) by a new mini-column affinity chromatography and its clinical value in diagnosis of hepatocellular carcinoma
Institute of Scientific and Technical Information of China (English)
Wei Wu; Dengfu Yao; Liwei Qiu; Xiaoxiao Gu; Xinhua Wu
Objective: To establish a convenient and economic method to determine hepatoma-specific α-fetoprotein (HS-AFP) for diagnosis of hepatocellular carcinoma (HCC). Methods: HS-AFP from serum of HCC patients was separated by a mini-column Lens culinaris agglutinin (LCA)-affinity chromatography. The levels of serum total AFP and separated HS-AFP were detected by radioimmunoassay (RIA). Results: Circulating AFP was separated into three peaks (AFP-1, AFP-2, and AFP-3) by LCA-affinity chromatography. During the elution course, the AFP-1 and AFP-2 could be eluted with TE buffer. HS-AFP (AFP-3) from sera of HCC patients was eluted dearly on the LCA-sepharose gel mini-column with a solution containing a-methyl-D-mannoside. It was a part of total AFP and only found in sera of HCC patients. A ratio of more than 15% for HS-AFP to total AFP in serum was considered as a specific marker for HCC diagnosis with higher sensitivity (92.7%) and specificity(88.2%). Conclusion: The new assay for circulating HS-AFP analysis is more sensitive, repeatable, and convenient. Its clinical application would be useful to early diagnosis of HCC.
The Carnegie-Spitzer-IMACS Redshift Survey of Galaxy Evolution since z=1.5: I. Description and Methodology and More!
Kelson, Daniel D; Dressler, Alan; McCarthy, Patrick J; Shectman, Stephen A; Mulchaey, John S; Villanueva, Edward V; Crane, Jeffrey D; Quadri, Ryan F
We describe the Carnegie-Spitzer-IMACS (CSI) Survey, a wide-field, near-IR selected spectrophotometric redshift survey with IMACS on Magellan-Baade. CSI uses a flux-limited sample of galaxies in Spitzer IRAC 3.6micron imaging of SWIRE fields to efficiently trace the stellar mass of average galaxies to z~1.5. This paper provides an overview of the survey selection, observations, and processing of the photometry and spectrophotometry. We also describe the analysis of the data: new methods of fitting synthetic SEDs are used to derive redshifts, stellar masses, emission line luminosities, and coarse information on recent star-formation. Our unique methodology for analyzing low-dispersion spectra taken with multilayer prisms in IMACS, combined with panchromatic photometry from the ultraviolet to the IR, has yielded high quality redshifts for 43,347 galaxies in our first 5.3 sq. degs of the SWIRE XMM-LSS field. A new approach to assessing data quality is also described, and three different approaches are used to es...
Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.
Piovesana, Susy; Capriotti, Anna Laura; Cavaliere, Chiara; Ferraris, Francesca; Samperi, Roberto; Ventura, Salvatore; Laganà, Aldo
Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides. PMID:26851086
Meyer, Mathieu; Schuett, Carsten; Werner, Elisabeth M.
An affine invariant point on the class of convex bodies in R^n, endowed with the Hausdorff metric, is a continuous map p which is invariant under one-to-one affine transformations A on R^n, that is, p(A(K))=A(p(K)). We define here the new notion of dual affine point q of an affine invariant point p by the formula q(K^{p(K)})=p(K) for every convex body K, where K^{p(K)} denotes the polar of K with respect to p(K). We investigate which affine invariant points do have a dual point, whether this ...
DEFF Research Database (Denmark)
Høholdt, Tom; Beelen, Peter; Ghorpade, Sudhir Ramakant
We consider a new class of linear codes, called affine Grassmann codes. These can be viewed as a variant of generalized Reed-Muller codes and are closely related to Grassmann codes.We determine the length, dimension, and the minimum distance of any affine Grassmann code. Moreover, we show that...... affine Grassmann codes have a large automorphism group and determine the number of minimum weight codewords....
Bioaffinity chromatography on monolithic supports
Tetala, K.K.R.; Beek, van T.A.
Affinity chromatography on monolithic supports is a powerful analytical chemical platform because it allows for fast analyses, small sample volumes, strong enrichment of trace biomarkers and applications in microchips. In this review, the recent research using monolithic materials in the field of bi
Multidimensional Strategy for Sensitive Phosphoproteomics Incorporating Protein Prefractionation Combined with SIMAC, HILIC, and TiO(2) Chromatography Applied to Proximal EGF Signaling
DEFF Research Database (Denmark)
Engholm-Keller, Kasper; Hansen, Thomas Aarup; Palmisano, Giuseppe; Larsen, Martin R
applied to 400 µg of protein from EGF stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into mono-phosphorylated and multi-phosphorylated pools using Sequential elution from...... IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and non-phosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4,700 unique phosphopeptides, while 636 phosphosites were changing...
A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry
Energy Technology Data Exchange (ETDEWEB)
Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F
Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid
Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin
Directory of Open Access Journals (Sweden)
Renata Angeli
Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.
Ferromagnetic levan composite: an affinity matrix to purify lectin.
Angeli, Renata; da Paz, Nathalia V N; Maciel, Jackeline C; Araújo, Flávia F B; Paiva, Patrícia M G; Calazans, Glícia M T; Valente, Ana Paula; Almeida, Fábio C L; Coelho, Luana C B B; Carvalho, Luiz B; Silva, Maria da Paz C; dos Santos Correia, Maria Tereza
A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column. PMID:19547713
Affine and Projective Geometry
Bennett, M K
An important new perspective on AFFINE AND PROJECTIVE GEOMETRY. This innovative book treats math majors and math education students to a fresh look at affine and projective geometry from algebraic, synthetic, and lattice theoretic points of view. Affine and Projective Geometry comes complete with ninety illustrations, and numerous examples and exercises, covering material for two semesters of upper-level undergraduate mathematics. The first part of the book deals with the correlation between synthetic geometry and linear algebra. In the second part, geometry is used to introduce lattice theory
Energy Technology Data Exchange (ETDEWEB)
Gueltekin, Kemal [Izmir Institute of Technology, Department of Physics, Izmir (Turkey)
In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schroedinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor. (orig.)
Affine and degenerate affine BMW algebras: Actions on tensor space
Daugherty, Zajj; Virk, Rahbar
The affine and degenerate affine Birman-Murakami-Wenzl (BMW) algebras arise naturally in the context of Schur-Weyl duality for orthogonal and symplectic quantum groups and Lie algebras, respectively. Cyclotomic BMW algebras, affine and cyclotomic Hecke algebras, and their degenerate versions are quotients. In this paper we explain how the affine and degenerate affine BMW algebras are tantalizers (tensor power centralizer algebras) by defining actions of the affine braid group and the degenerate affine braid algebra on tensor space and showing that, in important cases, these actions induce actions of the affine and degenerate affine BMW algebras. We then exploit the connection to quantum groups and Lie algebras to determine universal parameters for the affine and degenerate affine BMW algebras. Finally, we show that the universal parameters are central elements--the higher Casimir elements for orthogonal and symplectic enveloping algebras and quantum groups.
Optimizing TiO2-based phosphopeptide enrichment for automated multidimensional liquid chromatography coupled to tandem mass spectrometry
Cantin, Greg T.; Shock, Teresa R.; Park, Sung Kyu; Madhani, Hiten D.; Yates, John R.
An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO2. A comparison of TiO2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO2, was also carried out using trypt...
Karasek, Francis W.; And Others
This review covers fundamental developments in gas chromatography during 1982 and 1983. Literature is considered under these headings: columns; liguid phases; solid supports; sorption processes and solvents; open tubular column gas chromatography; instrumentation; high-resolution columns and applications; other techniques; qualitative and…
Single Bead Affinity Detection (SINBAD) for the Analysis of Protein-Protein Interactions
Schulte, Roberta; Talamas, Jessica; Doucet, Christine; Hetzer, Martin W.
We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform...
DEFF Research Database (Denmark)
Skjødt, Mette Louise
Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...
Affine and degenerate affine BMW algebras: The center
Daugherty, Zajj; Virk, Rahbar
The degenerate affine and affine BMW algebras arise naturally in the context of Schur-Weyl duality for orthogonal and symplectic Lie algebras and quantum groups, respectively. Cyclotomic BMW algebras, affine Hecke algebras, cyclotomic Hecke algebras, and their degenerate versions are quotients. In this paper the theory is unified by treating the orthogonal and symplectic cases simultaneously; we make an exact parallel between the degenerate affine and affine cases via a new algebra which takes the role of the affine braid group for the degenerate setting. A main result of this paper is an identification of the centers of the affine and degenerate affine BMW algebras in terms of rings of symmetric functions which satisfy a "cancellation property" or "wheel condition" (in the degenerate case, a reformulation of a result of Nazarov). Miraculously, these same rings also arise in Schubert calculus, as the cohomology and K-theory of isotropic Grassmanians and symplectic loop Grassmanians. We also establish new inte...
Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge
Hellman, Lance M.; Zhao, Chunxia; Melikishvili, Manana; Tao, Xiaorong; Hopper, James E.; Whiteheart, Sidney W.; Fried, Michael G.
Many recombinant proteins carry an oligohistidine (HisX)-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion–nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins an...
Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS
Akashi, Tetsuyuki; Yamori, Takao
Background Phosphoproteins play important roles in a vast series of biological processes. Recent proteomic technologies offer the comprehensive analyses of phosphoproteins. Recently, we demonstrated that surface-enhanced laser desorption/ionization time of flight mass (SELDI-TOF MS) would detect phosphoproteins quantitatively, which was a new application of SELDI-TOF MS. Results We combined immobilized metal affinity chromatography (IMAC) with SELDI-TOF MS. After SELDI-TOF MS analysis of IMAC...
Hierarchical Affinity Propagation
Givoni, Inmar; Frey, Brendan J
Affinity propagation is an exemplar-based clustering algorithm that finds a set of data-points that best exemplify the data, and associates each datapoint with one exemplar. We extend affinity propagation in a principled way to solve the hierarchical clustering problem, which arises in a variety of domains including biology, sensor networks and decision making in operational research. We derive an inference algorithm that operates by propagating information up and down the hierarchy, and is efficient despite the high-order potentials required for the graphical model formulation. We demonstrate that our method outperforms greedy techniques that cluster one layer at a time. We show that on an artificial dataset designed to mimic the HIV-strain mutation dynamics, our method outperforms related methods. For real HIV sequences, where the ground truth is not available, we show our method achieves better results, in terms of the underlying objective function, and show the results correspond meaningfully to geographi...
Affinity driven social networks
Ruyú, B.; Kuperman, M. N.
In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.
Affine General Equilibrium Models
Bjørn Eraker
No-arbitrage models are extremely flexible modelling tools but often lack economic motivation. This paper describes an equilibrium consumption-based CAPM framework based on Epstein-Zin preferences, which produces analytic pricing formulas for stocks and bonds under the assumption that macro growth rates follow affine processes. This allows the construction of equilibrium pricing formulas while maintaining the same flexibility of state dynamics as in no-arbitrage models. In demonstrating the a...
Gaussian Affine Feature Detector
Xu, Xiaopeng; Zhang, Xiaochun
A new method is proposed to get image features' geometric information. Using Gaussian as an input signal, a theoretical optimal solution to calculate feature's affine shape is proposed. Based on analytic result of a feature model, the method is different from conventional iterative approaches. From the model, feature's parameters such as position, orientation, background luminance, contrast, area and aspect ratio can be extracted. Tested with synthesized and benchmark data, the method achieve...
免疫亲和柱净化-高效液相色谱法同时检测小麦中脱氧雪腐镰刀菌烯醇及其衍生物%Simultaneous determination of deoxynivalenol and ramification in wheat by immune affinity column and high performance liquid chromatography
Institute of Scientific and Technical Information of China (English)
王培; 徐广超; 尹丽梅; 黎睿; 谢刚
建立脱氧雪腐镰刀菌烯醇(DON)及其衍生物3-乙酰基—脱氧雪腐镰刀菌烯醇(3-A-DON)和15-乙酰基—脱氧雪腐镰刀菌烯醇(15-A-DON)的免疫亲和柱净化—高效液相色谱同时检测方法。小麦样品经超纯水提取、免疫亲和柱净化、吹干、定容后,用配有紫外检测器的高效液相色谱仪进行DON、3-A-DON和15-A-DON三种毒素的同时检测,并与液相色谱质谱法比对。结果表明:该方法检出限,DON、3-A-DON和15-A-DON均为100μg/kg,样品加标回收率范围86.6%~96.5%,变异系数小于10%,具有较好的准确性和精密度。该方法简单、快速、灵敏度高、选择性好,适用于小麦中DON、3-A-DON和15-A-DON的同时检测。%The method to determine deoxynivalenol (DON),3-acetyl- deoxynivalenol (3-A-DON) and 15-acetyl-deoxynivalenol (15 -A -DON )in wheat simultaneously by immune affinity column clean-up and high performance liquid chromatography was established. The wheat samples were extrac-ted by ultrapure water,cleared up with the immuno-affinity column (IAC). After drying and constant volume,three toxins,DON、3-A-DON and15-A-DON,were detected simultaneously by high per-formance liquid chromatograph with UV detector,and compared with that by Liquid chromatography mass spectrometry. The result showed that the detection limit of method was 100μg/kg for DON,3-A-DON and 15-A-DON respectively. Recovery rates in wheat spiked with DON,3-A-DON,and 15-A-DON,ranged from 86. 6% to 96. 5%. The variation coefficient was less than 10%. This method was suitable for determination of DON,3 -A-DON,and 15 -A-DON in wheat,with the advantages of simplicity,rapidness,sensitivity and good selectivity,veracity and precision.
Purificación de IgG1, IgG2 e IgG3 mediante cromatografía de afinidad a partir de intacglobín Purification of IgG1, IgG2 and IgG3 by affinity chromatography starting from Intacglobin
Directory of Open Access Journals (Sweden)
Ada A Arce Hernández
Full Text Available Se describe un método de purificación de las subclases IgG1, IgG2 e IgG3 a partir de Intacglobin mediante cromatografía de afinidad con el empleo de proteína A Sepharose y la aplicación de un gradiente lineal de pH. Se detectó la presencia de IgG1, IgG2 e IgG3 en las fracciones eluidas mediante doble inmunodifusión e inmunoelectroforesis, utilizando sueros comerciales anti IgG, anti IgG1, anti IgG2 y anti IgG3. El procedimiento desarrollado se caracterizó por su sencillez, elevada resolución y la relativa pureza de las subclases aisladas. Se logró un rendimiento total del 53,7 %, lo que indica la eficiencia del métodoA method for the purification of IgG1, IgG2, and IgG3 subclasses starting from Intacglobin by affinity chromatography with the use of protein A Sepharose and the application of a lineal gradient of pH is described. IgG1, IgG2 and IgG3 were detected in the eluted fractions by doble immunodifussion and immunoelectrophoresis by using anti IgG, anti IgG1, anti IgG2 and anti IgG3 commercial sera. The procedure developed was characterized by its simplicity, high resolution and the relative purity of the isolated subclasses. A total yield of 53.7 % was attained, which demonstrates the efficiency of this method
Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis
Directory of Open Access Journals (Sweden)
Matthew P. Stokes
Full Text Available Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level. Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping. Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.
On the Affine Isoperimetric Inequalities
Indian Academy of Sciences (India)
Wuyang Yu; Gangsong Leng
We obtain an isoperimetric inequality which estimate the affine invariant -surface area measure on convex bodies. We also establish the reverse version of -Petty projection inequality and an affine isoperimetric inequality of $_{-p}K$.
Adjoint affine fusion and tadpoles
Urichuk, Andrew; Walton, Mark A.
We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-pol...
Gaussian Affine Feature Detector
Xu, Xiaopeng
A new method is proposed to get image features' geometric information. Using Gaussian as an input signal, a theoretical optimal solution to calculate feature's affine shape is proposed. Based on analytic result of a feature model, the method is different from conventional iterative approaches. From the model, feature's parameters such as position, orientation, background luminance, contrast, area and aspect ratio can be extracted. Tested with synthesized and benchmark data, the method achieves or outperforms existing approaches in term of accuracy, speed and stability. The method can detect small, long or thin objects precisely, and works well under general conditions, such as for low contrast, blurred or noisy images.
Mulik, James D.; Sawicki, Eugene
Accurate for the analysis of ions in solution, this form of analysis enables the analyst to directly assay many compounds that previously were difficult or impossible to analyze. The method is a combination of the methodologies of ion exchange, liquid chromatography, and conductimetric determination with eluant suppression. (Author/RE)
An affine framework for analytical mechanics
Urbanski, Pawel
An affine Cartan calculus is developed. The concepts of special affine bundles and special affine duality are introduced. The canonical isomorphisms, fundamental for Lagrangian and Hamiltonian formulations of the dynamics in the affine setting are proved.
The Protocol for Phosphoprotein Enrichment in Rice (Oryza sativa L.)——Phenol Extraction Combined with Immobilized Metal Affinity Chromatography%一种适用于双向电泳的水稻磷酸化蛋白富集方法——酚提取法结合固相金属离子亲和层析法
Institute of Scientific and Technical Information of China (English)
邓湘雄; 张晓勤; 胡江; 徐祥彬; 郭龙彪; 陈慧男; 应奇才; 王慧中; 薛大伟
磷蛋白在植物信号传导和胁迫响应中有着非常重要的作用,磷蛋白质组学研究已经成为蛋白质组学研究领域中备受瞩目的一个部分.本研究用酚提取法以水稻日本晴苗期叶片为材料提取叶片总蛋白,提取率达3.5%;用固相金属离子亲和层析柱纯化富集磷蛋白,得到磷蛋白占总蛋白约6.4%.对过柱洗涤液、不同阶段洗脱液等各个组分进行SDS-PAGE,粗略检测其蛋白含量,并根据单向SDS-PAGE结果对总蛋白、高峰段磷蛋白、非高峰段磷蛋白以及富集后再纯化的总磷蛋白进行双向电泳,比较其中的蛋白差异.本研究提出的方法和程序可在7 cm聚丙烯酰胺凝胶上检测到多达856个磷蛋白,是一种非常有效的磷蛋白富集、纯化和分离鉴定的方法.%Phosphoproteins play very important roles in plant signal transduction and stress responses. Phosphoproteomics has become a big concern for proteomic researchers. Total protein was extracted from rice (Oryza sativa L. Japonica nipponbare) leaves via phenol extraction in this study. Protein mass obtained is up to 3.5% in fresh rice leaves; phosphoprotein was enriched by an immobilized metal affinity chromatography column, which takes a proportion of 6. 4% in the total protein. Protein profiles of washing solution, elution solutions and others were approximately exhibited by SDS-PAGE. Based on the results, two-dimensional polyacrylamide gel eleclrophoresis (2D-PAGE) for total proteins, proteins from peak fraction, non-peak fractions and purified phosphoprotein after enrichment were carried out to better understand the variance among these samples on two dimensional gels. The protocol and procedure described here could dissociate as more as 856 spots per polyacrylamide gel of 7 cm. It is a highly effective way to separate and detecte phosphoproteins.
Adjoint affine fusion and tadpoles
Urichuk, Andrew; Walton, Mark A.
We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.
Adjoint affine fusion and tadpoles
Urichuk, Andrew
We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows, and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.
Energy Technology Data Exchange (ETDEWEB)
Tarter, J.G.
Similar in many ways to other liquid chromatographic techniques, ion chromatography (IC) is an invaluable method that is used all too rarely in chemical analysis. Its application is particularly promising in the analysis of inorganic anions, an area where more information is needed. Ion Chromatogrphy identifies the merits of this technique. Serving as a review and reference for experienced ion chromatographers and as a teaching aid for individuals new to the field, it provides the information necessary for determining the potential usefulness of IC for a given situation. Among the areas treated, this volume focuses on eluant-suppressed IC, especially for anion and cation analysis, single-column IC, plus ancillary techniques and applications, electrochemical, spectroscopic, and other detecting methods, ion chromatography exclusion (ICE), including its limits and applications.
Affinity approaches in RNAi-based therapeutics purification.
Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani
The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537
Preparation of Affinity Column Based on Zr{sup 4+} Ion for Phosphoproteins Isolation
Energy Technology Data Exchange (ETDEWEB)
Lee, Seon Mi; Bae, In Ae; Park, Jung Hyen; Kim, Tae Dong; Choi, Seong Ho [Hannam University, Daejeon (Korea, Republic of)
This paper has described about preparation of Zr{sup 4+} affinity column based on the poly(styreneco- glycidyl methacrylate) prepared by emulsion polymerization of styrene and glycidyl methacrylate in order to isolate phosphopeptide. The Zr{sup 4+} ions were introduced after the phophonation of an epoxy group on polymeric microspheres. The successful preparation of Zr{sup 4+}-immobilized polymeric microsphere stationary phase was confirmed through Fourier transform infrared spectra, optical microscopy, scanning electron microscopy, X-ray photoelectron spectra and inductively coupled plasma-atomic emission spectrometer. The separation efficiency for Zr{sup 4+} affinity column prepared by slurry packing was tested to phosphonated casein and dephosphonated casein. The resolution time (min) of the phosphonated casein was higher than that of dephosphated casein for Zr{sup 4+} affinity polymeric microsphere by liquid chromatography. This Zr{sup 4+} affinity column can be used for isolation of phosphonated casein from casein using liquid chromatography.
Preparation of Affinity Column Based on Zr4+ Ion for Phosphoproteins Isolation
International Nuclear Information System (INIS)
This paper has described about preparation of Zr4+ affinity column based on the poly(styreneco- glycidyl methacrylate) prepared by emulsion polymerization of styrene and glycidyl methacrylate in order to isolate phosphopeptide. The Zr4+ ions were introduced after the phophonation of an epoxy group on polymeric microspheres. The successful preparation of Zr4+-immobilized polymeric microsphere stationary phase was confirmed through Fourier transform infrared spectra, optical microscopy, scanning electron microscopy, X-ray photoelectron spectra and inductively coupled plasma-atomic emission spectrometer. The separation efficiency for Zr4+ affinity column prepared by slurry packing was tested to phosphonated casein and dephosphonated casein. The resolution time (min) of the phosphonated casein was higher than that of dephosphated casein for Zr4+ affinity polymeric microsphere by liquid chromatography. This Zr4+ affinity column can be used for isolation of phosphonated casein from casein using liquid chromatography
Purification of recombinant aprotinin produced in transgenic corn seed: separation from CTI utilizing ion-exchange chromatography
Directory of Open Access Journals (Sweden)
A. R. Azzoni
Full Text Available Protein expression in transgenic plants is considered one of the most promising approaches for producing pharmaceutical proteins. As has happened with other recombinant protein production schemes, the downstream processing (dsp of these proteins produced in plants is key to the technical and economic success of large-scale applications. Since dsp of proteins produced transgenically in plants has not been extensively studied, it is necessary to broaden the investigation in this field in order to more precisely evaluate the commercial feasibility of this route of expression. In this work, we studied the substitution of an IMAC chromatographic step, described in previous work (Azzoni et al., 2002, with ion-exchange chromatography on SP Sepharose Fast Flow resin as the second step in the purification of recombinant aprotinin from transgenic maize seed. The main goal of this second purification step is to separate the recombinant aprotinin from the native corn trypsin inhibitor. Analysis of the adsorption isotherms determined at 25°C under different conditions allowed selection of 0.020 M Tris pH 8.5 as the adsorption buffer. The cation-exchange chromatographic process produced a high-purity aprotinin that was more than ten times more concentrated than that generated using an IMAC step.
Titanium dioxide as chemo-affinity chromatographic sorbent of biomolecular compounds - Applications in acidic modification-specific proteomics
DEFF Research Database (Denmark)
Engholm-Keller, Kasper; Larsen, Martin R
matrices for further characterization is affinity chromatography, which relies on the specific interaction between an analyte in solution and a solid adsorbent. Titanium dioxide-based affinity chromatography has proven to be a versatile tool in enrichment of various compounds such as phosphorylated...... biomolecules due to its unique ion and ligand exchange properties and high stability towards pH and temperature. Recently, titanium dioxide chromatography was introduced in proteomics as a highly specific method for enriching phosphorylated peptides - a method, which has been widely adapted by the field of...
Infinite transitivity on affine varieties
Arzhantsev, Ivan; Flenner, Hubert; Kaliman, Shulim; Kutzschebauch, Frank; ZAIDENBERG, MIKHAIL
In this note we survey recent results on automorphisms of affine algebraic varieties, infinitely transitive group actions and flexibility. We present related constructions and examples, and discuss geometric applications and open problems.
Representations of affine Hecke algebras
Xi, Nanhua
Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest
International Nuclear Information System (INIS)
In the theory of affine SL(2)-embeddings, which was constructed in 1973 by Popov, a locally transitive action of the group SL(2) on a normal affine three-dimensional variety X is determined by a pair (p/q,r), where 0GV//T-hat. In the substantiation of this result a key role is played by Cox's construction in toric geometry. Bibliography: 12 titles
Cromatografia unificada Unified chromatography
Directory of Open Access Journals (Sweden)
Carin von Mühlen
Full Text Available The scope of this study encompasses an overview of the principles of unified chromatography as well as the principles of chromatographic techniques as applied to unified systems, which include gas chromatography, liquid chromatography, supercritical fluid chromatography, high temperature and high pressure liquid chromatography, micro-liquid chromatography, enhanced fluidity chromatography, and solvating gas chromatography. Theoretical considerations and individual instrumental parameters such as mobile phase, sample introduction system, columns, and detection system are also discussed. Future applications of this separation approach are discussed.
Electrochemical affinity biosensors for detection of mycotoxins: A review.
Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R
This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. PMID:23743326
Affine density in wavelet analysis
Kutyniok, Gitta
In wavelet analysis, irregular wavelet frames have recently come to the forefront of current research due to questions concerning the robustness and stability of wavelet algorithms. A major difficulty in the study of these systems is the highly sensitive interplay between geometric properties of a sequence of time-scale indices and frame properties of the associated wavelet systems. This volume provides the first thorough and comprehensive treatment of irregular wavelet frames by introducing and employing a new notion of affine density as a highly effective tool for examining the geometry of sequences of time-scale indices. Many of the results are new and published for the first time. Topics include: qualitative and quantitative density conditions for existence of irregular wavelet frames, non-existence of irregular co-affine frames, the Nyquist phenomenon for wavelet systems, and approximation properties of irregular wavelet frames.
Inhomogeneous self-affine carpets
Fraser, Jonathan M.
We investigate the dimension theory of inhomogeneous self-affine carpets. Through the work of Olsen, Snigireva and Fraser, the dimension theory of inhomogeneous self-similar sets is now relatively well-understood, however, almost no progress has been made concerning more general non-conformal inhomogeneous attractors. If a dimension is countably stable, then the results are immediate and so we focus on the upper and lower box dimensions and compute these explicitly for large classes of inhomo...
A Comparative Study of Lectin Affinity Based Plant N-Glycoproteome Profiling Using Tomato Fruit as a Model*
Ruiz-May, Eliel; Hucko, Simon; Kevin J. Howe; Zhang, Sheng; Sherwood, Robert W.; Thannhauser, Theodore W; Rose, Jocelyn K. C.
Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with different affinities can be particularly effective. A multi-lectin approach has also been reported to provide a significant benefit for the analysis of plant N-glycoproteins; however, it has yet to be de...
Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae
Fang, Lin; Gan, Zhibo; Marquardt, Ronald R.
An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac+ fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound recept...
Alternative affinity tools: more attractive than antibodies?
Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.
Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids
Spectral affinity in protein networks
Directory of Open Access Journals (Sweden)
Teng Shang-Hua
Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to
Crystal structures of fusion proteins with large-affinity tags.
Smyth, Douglas R; Mrozkiewicz, Marek K; McGrath, William J; Listwan, Pawel; Kobe, Bostjan
The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. PMID:12824478
Con A affinity glycoproteomics of normal human liver tissue
Institute of Scientific and Technical Information of China (English)
In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were gener- ated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence stain- ing based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were de- tected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF- MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.
Manifolds with integrable affine shape operator
Directory of Open Access Journals (Sweden)
Daniel A. Joaquín
Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.
Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening
Institute of Scientific and Technical Information of China (English)
张虹; 潘远江
A method of screening assay is demonstrated. The approach is based on the affinity ofantitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concentration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detection (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.
Affinity ultrafiltration of DNA topoisomerases-targeted compounds determined with HPLC/ESI-MS for drug candidate screening
Institute of Scientific and Technical Information of China (English)
张虹; 潘远江
A method of screening assay is demonstrated. The approach is based on the affinity of antitumor candidates for topoisomerases. In this method, antitumor candidates are fished out using topoisomerases as targets. Traditional analysis of complex compounds typically encounters signal suppression due to the relatively low concentrations, but enzyme-affinity screening for the active compounds can effectively concentrate the desired analysts into a small volume of high concen-tration. Active compounds are separated from non-affinity compounds by ultrafiltration. The molecules-enzymes complexes that are retained on the filter are subsequently separated by acidification to obtain the topoisomerases-affinity compounds for analysis on High Performance Liquid Chromatography coupled with electrospray ionization mass spectrometric detec-tion (ESI-MS). This enzyme-affinity based screening assay provides a highly specific and efficient method that can directly screen, identify, and acquire drug candidates thus improving the accuracy and speed of high-throughput screening activities.
Improved Thermal Modulator for Gas Chromatography
Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.
An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.
Steiner, Wolfgang
An integral self-affine tile is the solution of a set equation $\\mathbf{A} \\mathcal{T} = \\bigcup_{d \\in \\mathcal{D}} (\\mathcal{T} + d)$, where $\\mathbf{A}$ is an $n \\times n$ integer matrix and $\\mathcal{D}$ is a finite subset of $\\mathbb{Z}^n$. In the recent decades, these objects and the induced tilings have been studied systematically. We extend this theory to matrices $\\mathbf{A} \\in \\mathbb{Q}^{n \\times n}$. We define rational self-affine tiles as compact subsets of the open subring $\\mathbb{R}^n\\times \\prod_\\mathfrak{p} K_\\mathfrak{p}$ of the ad\\'ele ring $\\mathbb{A}_K$, where the factors of the (finite) product are certain $\\mathfrak{p}$-adic completions of a number field $K$ that is defined in terms of the characteristic polynomial of $\\mathbf{A}$. Employing methods from classical algebraic number theory, Fourier analysis in number fields, and results on zero sets of transfer operators, we establish a general tiling theorem for these tiles. We also associate a second kind of tiles with a rational matr...
The affine quantum gravity programme
International Nuclear Information System (INIS)
The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix { g-hat ab(x)} composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that still retain some basic characteristics of gravity, specifically a partial second-class constraint operator structure. Although perturbatively nonrenormalizable, gravity may possibly be understood nonperturbatively from a hard-core perspective that has proved valuable for specialized models. Finally, developing a procedure to pass to the genuine physical Hilbert space involves several interconnected steps that require careful coordination
Protein digestion and phosphopeptide enrichment on a glass microchip
Yue, Guihua Eileen; Roper, Michael G.; Balchunas, Catherine; Pulsipher, Abigail; Coon, Joshua J.; Shabanowitz, Jeffery; Hunt, Donald F.; Landers, James P.; Ferrance, Jerome P.
This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized t...
Fun with Paper Chromatography.
Coleman, Dava; Hounshell, Paul B.
Discusses paper chromatographic techniques and provides examples of typical classroom activities. Includes description of retardation values obtained during chromatography exercises and suggests using them for math lessons. (JN)
A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model
Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with differ...
Conformal field theory on affine Lie groups
International Nuclear Information System (INIS)
Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs
Conformal field theory on affine Lie groups
Energy Technology Data Exchange (ETDEWEB)
Clubok, K.S.
Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.
Maximin affinity learning of image segmentation
Turaga, Srinivas C; Helmstaedter, Moritz; Denk, Winfried; Seung, H Sebastian
Images can be segmented by first using a classifier to predict an affinity graph that reflects the degree to which image pixels must be grouped together and then partitioning the graph to yield a segmentation. Machine learning has been applied to the affinity classifier to produce affinity graphs that are good in the sense of minimizing edge misclassification rates. However, this error measure is only indirectly related to the quality of segmentations produced by ultimately partitioning the affinity graph. We present the first machine learning algorithm for training a classifier to produce affinity graphs that are good in the sense of producing segmentations that directly minimize the Rand index, a well known segmentation performance measure. The Rand index measures segmentation performance by quantifying the classification of the connectivity of image pixel pairs after segmentation. By using the simple graph partitioning algorithm of finding the connected components of the thresholded affinity graph, we are ...
A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.
Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco
Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification). PMID:27279446
Moringa oleifera lectin and binding affinity to humic acids : application to water treatment
Santos, Andréa F. S.; Luz, Luciana A.; Cunha, M. G. C.; Paiva, Patrícia M.G.; Teixeira, J.A.; Brito, A. G.; R. Nogueira; L. C. B. B. Coelho
Humic acids, natural organic matter, constitute a problem in water treatment; they react with chlorine forming undesirable disinfection byproducts. The aim of this work was the purification of a saline soluble Moringa oleifera lectin (SSMoL) and characterization of its affinity to humic acids. SSMoL isolation included extraction of seed flour, saline fractionation and guar gel chromatography. Hemagglutinating activity (HA) of extract (E), fraction (0-60F) and SSMoL was assessed...
Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin
White, Jim F.; Reinhard Grisshammer
BACKGROUND: Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram q...
Affinity capillary electrophoresis and density functional theory employed for characterization of (bio)molecular interactions
Czech Academy of Sciences Publication Activity Database
Kašička, Václav; Ehala, Sille; Růžička, Martin; Dybal, Jiří; Toman, Petr
Salzburg: Society of Analytical Chemistry, 2014. OR28. [ISC 2014. International Symposium on Chromatography /30./. 14.09.2014-18.09.2014, Salzburg] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S Institutional support: RVO:61388963 ; RVO:61389013 Keywords : affinity capillary electrophoresis * density functional theory * biomolecular complexes Subject RIV: CB - Analytical Chemistry, Separation
Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin
White, Jim F.; Grisshammer, Reinhard
Background Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram qu...
Methods for Improving Aptamer Binding Affinity
Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro
Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of a...
Covariant Functional Calculi from the Affine Groups
Gong, Yafang
Invoking the Clifford-Hermite Wavelets from Clifford analysis, we use the covariances of affine groups to construct a kind of functional calculi for several non-commuting bounded operators. Functional calculi are the intertwining transforms between the representations of affine groups in the space $L^2(\\mathbb R^m)$ and in the space of bounded operators. It turns out that the Weyl calculus is the value of this new functional calculus at the identity of affine groups. Our app...
Multipole solutions in metric-affine gravity
Socorro, J; Macías, A; Mielke, E W; Socorro, José; Lämmerzahl, Claus; Macías, Alfredo; Mielke, Eckehard W.
Above Planck energies, the spacetime might become non--Riemannian, as it is known fron string theory and inflation. Then geometries arise in which nonmetricity and torsion appear as field strengths, side by side with curvature. By gauging the affine group, a metric affine gauge theory emerges as dynamical framework. Here, by using the harmonic map ansatz, a new class of multipole like solutions in the metric affine gravity theory (MAG) is obtained.
Maximin affinity learning of image segmentation
Srinivas C Turaga; Briggman, Kevin L; Helmstaedter, Moritz; Denk, Winfried; Seung, H. Sebastian
Images can be segmented by first using a classifier to predict an affinity graph that reflects the degree to which image pixels must be grouped together and then partitioning the graph to yield a segmentation. Machine learning has been applied to the affinity classifier to produce affinity graphs that are good in the sense of minimizing edge misclassification rates. However, this error measure is only indirectly related to the quality of segmentations produced by ultimately partitioning the a...
Discrete Affine Minimal Surfaces with Indefinite Metric
Craizer, Marcos; Anciaux, Henri; Lewiner, Thomas
Inspired by the Weierstrass representation of smooth affine minimal surfaces with indefinite metric, we propose a constructive process producing a large class of discrete surfaces that we call discrete affine minimal surfaces. We show that they are critical points of an affine area functional defined on the space of quadrangular discrete surfaces. The construction makes use of asymptotic coordinates and allows defining the discrete analogs of some differential geometric objects, such as the n...
A Novel Vertex Affinity for Community Detection
Energy Technology Data Exchange (ETDEWEB)
Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.
Compact noncontraction semigroups of affine operators
Voynov, A. S.; Protasov, V. Yu
We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.
Protein Complex Purification by Affinity Capture.
LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P
Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601
Structural determinants of sigma receptor affinity
International Nuclear Information System (INIS)
The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-[3H]3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent
Structural determinants of sigma receptor affinity
Energy Technology Data Exchange (ETDEWEB)
Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.
The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.
Liquid Chromatography in 1982.
Freeman, David H.
Reviews trends in liquid chromatography including apparatus, factors affecting efficient separation of a mixture (peak sharpness and speed), simplified problem-solving, adsorption, bonded phase chromatography, ion selectivity, and size exclusion. The current trend is to control chemical selectivity by the liquid phase. (Author/JN)
Structure of classical affine and classical affine fractional W-algebras
International Nuclear Information System (INIS)
We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel'd and Sokolov reduction
Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.
Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh
Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a
Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.
Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan
Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV. PMID:24976378
Scaling analysis of affinity propagation.
Furtlehner, Cyril; Sebag, Michèle; Zhang, Xiangliang
We analyze and exploit some scaling properties of the affinity propagation (AP) clustering algorithm proposed by Frey and Dueck [Science 315, 972 (2007)]. Following a divide and conquer strategy we setup an exact renormalization-based approach to address the question of clustering consistency, in particular, how many cluster are present in a given data set. We first observe that the divide and conquer strategy, used on a large data set hierarchically reduces the complexity O(N2) to O(N((h+2)/(h+1))) , for a data set of size N and a depth h of the hierarchical strategy. For a data set embedded in a d -dimensional space, we show that this is obtained without notably damaging the precision except in dimension d=2 . In fact, for d larger than 2 the relative loss in precision scales such as N((2-d)/(h+1)d). Finally, under some conditions we observe that there is a value s* of the penalty coefficient, a free parameter used to fix the number of clusters, which separates a fragmentation phase (for ss*) of the underlying hidden cluster structure. At this precise point holds a self-similarity property which can be exploited by the hierarchical strategy to actually locate its position, as a result of an exact decimation procedure. From this observation, a strategy based on AP can be defined to find out how many clusters are present in a given data set. PMID:20866473
Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity.
Arendt, R M; Greenblatt, D J; Liebisch, D C; Luu, M D; Paul, S M
Factors influencing brain uptake of benzodiazepine derivatives were evaluated in adult Sprague Dawley rats (n = 8-10 per drug). Animals received single intraperitoneal doses of alprazolam, triazolam, lorazepam, flunitrazepam, diazepam, midazolam, desmethyldiazepam, or clobazam. Concentrations of each drug (and metabolites) in whole brain and serum 1 h after dosage were determined by gas chromatography. Serum free fraction was measured by equilibrium dialysis. In vitro binding affinity (apparent Ki) of each compound was estimated based on displacement of tritiated flunitrazepam in washed membrane preparations from rat cerebral cortex. Lipid solubility of each benzodiazepine was estimated using the reverse-phase liquid chromatographic (HPLC) retention index at physiologic pH. There was no significant relation between brain:total serum concentration ratio and either HPLC retention (r = 0.18) or binding Ki (r = -0.34). Correction of uptake ratios for free as opposed to total serum concentration yielded a highly significant correlation with HPLC retention (r = 0.78, P less than 0.005). However, even the corrected ratio was not correlated with binding Ki (r = -0.22). Thus a benzodiazepine's capacity to diffuse from systemic blood into brain tissue is much more closely associated with the physicochemical property of lipid solubility than with specific affinity. Unbound rather than total serum or plasma concentration most accurately reflects the quantity of drug available for diffusion. PMID:2888155
Free C+ actions on affine threefolds
Kraft, H.
We study algebraic actions of the additive group C+ on an affine threefold X and prove a smoothness property for the quotient morphism X -< X//C+. Then, following Shulim Kaliman, we give a proof of the conjecture that every free C+ action on affine 3-space C^3 is a translation.
Lectures on extended affine Lie algebras
Neher, Erhard
We give an introduction to the structure theory of extended affine Lie algebras, which provide a common framework for finite-dimensional semisimple, affine and toroidal Lie algebras. The notes are based on a lecture series given during the Fields Institute summer school at the University of Ottawa in June 2009.
Affine Constellations Without Mutually Unbiased Counterparts
Weigert, Stefan
It has been conjectured that a complete set of mutually unbiased bases in a space of dimension d exists if and only if there is an affine plane of order d. We introduce affine constellations and compare their existence properties with those of mutually unbiased constellations, mostly in dimension six. The observed discrepancies make a deeper relation between the two existence problems unlikely.
Affine constellations without mutually unbiased counterparts
Energy Technology Data Exchange (ETDEWEB)
Weigert, Stefan [Department of Mathematics, University of York, York YO10 5DD (United Kingdom); Durt, Thomas, E-mail: firstname.lastname@example.com, E-mail: firstname.lastname@example.com [IR-TONA, VUB, BE-1050 Brussels (Belgium)
It has been conjectured that a complete set of mutually unbiased bases in a space of dimension d exists if and only if there is an affine plane of order d. We introduce affine constellations and compare their existence properties with those of mutually unbiased constellations. The observed discrepancies make a deeper relation between the two existence problems unlikely. (fast track communication)
Dyes with high affinity for polylactide
Institute of Scientific and Technical Information of China (English)
Liang He; Shu Fen Zhang; Bing Tao Tang; Li Li Wang; Jin Zong Yang
Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes.The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.
Institute of Scientific and Technical Information of China (English)
Lifeng XI
In this paper,it is proved that any self-affine set satisfying the strong separation condition is uniformly porous.The author constructs a self-affine set which is not porous,although the open set condition holds.Besides,the author also gives a C1 iterated function system such that its invariant set is not porous.
Affine constellations without mutually unbiased counterparts
International Nuclear Information System (INIS)
It has been conjectured that a complete set of mutually unbiased bases in a space of dimension d exists if and only if there is an affine plane of order d. We introduce affine constellations and compare their existence properties with those of mutually unbiased constellations. The observed discrepancies make a deeper relation between the two existence problems unlikely. (fast track communication)
Improving image segmentation by learning region affinities
Energy Technology Data Exchange (ETDEWEB)
Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.
We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.
Faure, Karine
Abstract Liquid chromatography is one of the most powerful separation techniques as illustrated by its leading role in analytical sciences through both academic and industrial communities. Its implementation in microsystems appears to be crucial in the development of ?TAS. If electrophoretic techniques have been widely used in miniaturized devices, liquid chromatography has faced multiple challenges in the downsizing process. During the past five years significant breakthroughs...
Akapo, S. O.; Dimandja, J. M.; Kojiro, D. R.; Valentin, J. R.; Carle, G. C.
Gas chromatography has proven to be a very useful analytical technique for in situ analysis of extraterrestrial environments as demonstrated by its successful operation on spacecraft missions to Mars and Venus. The technique is also one of the six scientific instruments aboard the Huygens probe to explore Titan's atmosphere and surface. A review of gas chromatography in previous space missions and some recent developments in the current environment of fiscal constraints and payload size limitations are presented.
Optimized Affinity Capture of Yeast Protein Complexes.
LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P
Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596
Affinity Proteomics in the mountains: Alpbach 2015.
Taussig, Michael J
The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167
Corner Transfer Matrices and Quantum Affine Algebras
Foda, O E; Foda, Omar; Miwa, Tetsuji
Let H be the corner-transfer-matrix Hamiltonian for the six-vertex model in the anti-ferroelectric regime. It acts on the infinite tensor product W = V . V . V ....., where is the 2-dimensional irreducible representation of the quantum affine sl(2). We observe that H is the derivation of quantum affine sl(2), and conjecture that the eigenvectors of H form the level-1 vacuum representation of quantum affine sl(2). We report on checks in support of our conjecture.
Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin
Energy Technology Data Exchange (ETDEWEB)
Biessen, E.A.L; Horn, A.S.; Robillard, G.T. (Univ. of Groningen (Netherlands))
This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.
超高效液相色谱法快速检测粮食中黄曲霉毒素的含量%Rapid Analysis of Aflatoxins (B1, B2, G1, G2) in Grain by Immuno-affinity Clear-up Column and Ultra Performance Liquid Chromatography without Derivation
Institute of Scientific and Technical Information of China (English)
谢刚; 王松雪; 张艳
建立了免疫亲和柱净化-超高效液相色谱法快速测定粮食中黄曲霉毒素(Aflatoxins,AF)的检测方法.样品经提取后,用免疫亲和柱净化、浓缩,Waters Acquity UPLC BEH C18色谱柱(50 mm×2.1 mm,1.7 um)分离.以甲醇-水(40∶60,V/V)为流动相,流速为0.2 mL/min,进样量为1μL,荧光检测器检测,激发波长为360 nm,发射波长为440 nm,无需衍生.黄曲霉毒素B1,B2,G1,G2的保留时间小于5min,从样品前处理到结果分析整个过程小于45 min.根据3倍信噪比的峰响应值,确定黄曲霉毒素(B1,B2,G1,G2)检出限分别为0.15,0.05,0.40,0.06 pg,4种毒素在0.4 ~ 60.0 pg,0.2~15.0 pg,1.5~ 60.0 pg和0.2~15.0 pg范围内分别呈线性相关,相关系数R2值分别为0.9999,0.9999,0.9998和0.9992;在小麦、玉米、稻谷3类样品中加标回收率为77.4% ~ 104.2%,精密度为1.8% ~ 8.9%.本方法无需衍生即可同时测定粮食中4种黄曲霉毒素,适用于粮食中黄曲霉毒素的快速定量测定.%A rapid and environmental-friendly analytical method without any derivation was established for the determination of aflatoxins (B1, B2, G1, G2) in grain samples. The samples were extracted by methanol-water, cleared up with the immuno-affinity column (IAC). The separation of target compound was performed on a Waters Acquity UPLC BEH C18(50 mmx2. 1 mm, 1. 7μ,m) using methanol; water (40 = 60, V/V) as mobile phase with a flow rate of 0. 2 mL/min at 25 ℃. The injection volume was 1 μL and detection wavelengths were set at 360 nm (λem) and 440 nm (λeX) using fluorescence detector (FLD). The retention time was less than 5 min, and the whole analytical time was less than 45 min. The detection limits of AFB1, AFB2, AFG1, and AFG2 were 0.15, 0.05, 0.40 and 0.06 pg, respectively. The linear detection ranges of AFB1, AFB2, AFG1, and AFG2 were 0. 4-60. 0, 0. 2-15. 0, 1. 5-60, 0. 2-15. 00 pg with correlation coefficients (R2) of 0.9999, 0.9999, 0.9998, 0.9992, respectively
Automorphisms in Birational and Affine Geometry
Ciliberto, Ciro; Flenner, Hubert; McKernan, James; Prokhorov, Yuri; Zaidenberg, Mikhail
The main focus of this volume is on the problem of describing the automorphism groups of affine and projective varieties, a classical subject in algebraic geometry where, in both cases, the automorphism group is often infinite dimensional. The collection covers a wide range of topics and is intended for researchers in the fields of classical algebraic geometry and birational geometry (Cremona groups) as well as affine geometry with an emphasis on algebraic group actions and automorphism groups. It presents original research and surveys and provides a valuable overview of the current state of the art in these topics. Bringing together specialists from projective, birational algebraic geometry and affine and complex algebraic geometry, including Mori theory and algebraic group actions, this book is the result of ensuing talks and discussions from the conference "Groups of Automorphisms in Birational and Affine Geometry" held in October 2012, at the CIRM, Levico Terme, Italy. The talks at the conference high...
Synthesis of a New Series of Bone Affinity Compounds
Institute of Scientific and Technical Information of China (English)
A new series of bone affinity compounds were synthesized by linking chrysophanol with 5-fluorouracil derivatives. Their bone affinity was established by hydroxyapafive (HA)affinity experiment in vitro, and their cytostatic effects were shown by the MTT assay.
Affine Moment Invariants Generated by Graph Method
Czech Academy of Sciences Publication Activity Database
Suk, Tomáš; Flusser, Jan
Roč. 44, č. 9 (2011), 2047 – 2056. ISSN 0031-3203 R&D Projects: GA ČR(CZ) GA102/08/1593 Institutional research plan: CEZ:AV0Z10750506 Keywords : Image moments * Object recognition * Affine transformation * Affine moment invariants * Pseudoinvariants * Graph representation * Irreducibility * Independence Subject RIV: IN - Informatics, Computer Science Impact factor: 2.292, year: 2011 http://library.utia.cas.cz/separaty/2011/ZOI/suk-0359752.pdf
On Affine Fusion and the Phase Model
Walton, Mark A.
A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the $su(n)$ Wess-Zumino-Novikov-Witten (WZNW) conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connec...
Purely affine elementary su(N) fusions
Rasmussen, Jorgen; Walton, Mark A.
We consider three-point couplings in simple Lie algebras -- singlets in triple tensor products of their integrable highest weight representations. A coupling can be expressed as a linear combination of products of finitely many elementary couplings. This carries over to affine fusion, the fusion of Wess-Zumino-Witten conformal field theories, where the expressions are in terms of elementary fusions. In the case of su(4) it has been observed that there is a purely affine elementary fusion, i.e...