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Click-iT labeling technology employs a bioorthogonal reactive chemistry for the in situ labeling of specific molecular populations, such as proteins that have been newly synthesized or post-translationally modified in some experimental time window of interest. The Click-iT labeling reaction is based on a copper-catalyzed azide–alkyne cycloaddition and derives its high degree of specificity from the fact that the azide and alkyne reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms.
Application of this reaction to in situ labeling of cells is a two-step process. First, one reaction partner—either an azide or alkyne linked to a "building block" such as an amino acid, monosaccharide, fatty acid, nucleotide or nucleoside—is biosynthetically incorporated. Subsequently, the other reaction partner—the complementary alkyne or azide linked to a fluorescent dye, biotin or other detection reagent—is "clicked" into place in the presence of catalytic copper (I), providing a detection moiety (Figure 9.4.1). One reaction partner must be an azide derivative and the other an alkyne derivative, but either functional moiety can serve as the biosynthetically incorporated molecule or the detection molecule (e.g., L-azidohomoalanine (AHA) + Alexa Fluor 488 alkyne is the inverse of the reaction scheme shown in Figure 9.4.1A).
The small size of alkyne and azide tags allows the biosynthetic building blocks to which they are attached to be processed by enzymes, such as aminoacyl tRNA synthetases and nucleotide polymerases, that have poor tolerance for substrates with larger modifications such as fluorescent organic dyes. Furthermore, the 1,2,3-triazole linkage between the azide and alkyne reaction partners (Figure 9.4.1) is extremely stable. It is not susceptible to hydrolysis, oxidation or reduction, and it survives ionization in mass spectrometry (MS) analysis. Click-iT labeling technology and the details of the click reaction are discussed in Click Chemistry—Section 3.1. For a complete list of azide and alkyne derivatives compatible with Click-iT labeling technology, see Molecular Probes azide and alkyne derivatives—Table 3.1, Click Chemistry—Section 3.1. Here we highlight the azide and alkyne derivatives that can be used for labeling newly synthesized proteins and detecting post-translational protein modifications.
The Click-iT AHA (L-azidohomoalanine, C10102) and Click-iT HPG (L-homopropargylglycine, C10186; Figure 9.4.1A) reagents are methionine surrogates that provide nonradioactive alternatives to 35S-methionine for pulse-chase detection of protein synthesis and degradation. These amino acid analogs are fed to cultured cells and incorporated into proteins during active protein synthesis. The enzymatically incorporated Click-iT AHA or Click-iT HPG is then detected with a fluorescent alkyne or fluorescent azide, respectively, using a Cu(I)-catalyzed click reaction. These Click-iT reagents provide detection sensitivity comparable to that obtained using the radioactive 35S-methionine method and are compatible with downstream LC-MS/MS and MALDI MS analysis, as well as with total-protein, glycoprotein and phosphoprotein gel stains for differential analyses of newly synthesized protein together with post-translational modifications.
Click-iT AHA is also available in the Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay Kit (C10289), which provides Alexa Fluor 488 alkyne for detection. Click-iT AHA has proven to be a successful substitute for methionine in many cell types, including COS-7, 3T3-L1, HeLa, HEK 293 and Jurkat cells. Note that cells should be labeled in methionine-free media, as methionine is the preferred substrate for methionyl tRNA transferase, and supplemented media (i.e., methionine-free DMEM) should be used in place of HBSS to achieve greater Click-iT AHA incorporation at lower concentrations.
The Click-iT metabolic glycoprotein labeling reagents provide biosynthetic precursors for detecting and characterizing post-translational glycosylation of proteins. Four azide- or alkyne-modified monosaccharides are available for metabolic incorporation into a specific subclass of protein glycan structures:
Cultured cells are simply incubated with the modified sugars for 2–3 days or until cells reach the appropriate density. The acetyl groups improve cell permeability of the modified sugars and are removed by nonspecific intracellular esterases (Figure 9.4.2). The resulting azide- or alkyne-modified sugar is then metabolically incorporated through the permissive nature of the oligosaccharide biosynthesis pathway, yielding functionalized glycoproteins that can be chemoselectively coupled to complementary alkyne- or azide-functionalized fluorophores and biotinylation reagents for detection or affinity capture. We offer three Click-iT Protein Analysis Detection Kits (C33370, C33371, C33372) described below for the detection of azide-functionalized glycoproteins in 1D or 2D electrophoresis gels or western blots. These labeled glycoproteins are compatible with total-protein, glycoprotein and phosphoprotein gel stains and provide a detection sensitivity of a few hundred femtomoles, allowing an in-depth analysis of low-abundance glycoproteins as well as glycoproteins with a small degree of glycosylation.
Glycoproteins labeled with the Click-iT labeling and detection reagents are also compatible with downstream LC-MS/MS and MALDI-MS analyses for further identification and characterization. For added convenience, we offer an O-GlcNAc peptide LC/MS standard (C33374) from the transcription factor CREB for LC-MS/MS and MALDI-MS analyses of the O-GlcNAc posttranslational modification. This peptide is also available together with its phosphorylated counterpart for use as LC/MS standards (C33373) in differential mass spectrometry–based studies of the corresponding modifications, as well as for characterizing differential β-elimination/addition conditions.
We also offer the the Click-iT O-GlcNAc Enzymatic Labeling System for in vitro enzyme-mediated N-azidoacetylgalactosamine labeling of O-GlcNAc–modified glycoproteins (C33368). Proteins are enzymatically labeled using the permissive mutant β-1,4-galactosyltransferase (Gal-T1, Y289L), which transfers azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues on the target proteins. Target proteins can then be detected using an alkyne-derivatized fluorophore or one of the Click-iT Protein Analysis Detection Kits described below. Using the Click-iT O-GlcNAc Enzymatic Labeling System in conjunction with the Click-iT Tetramethylrhodamine (TAMRA) Protein Analysis Detection Kit, we have detected as little as 1 picomole of α-crystallin, a protein which is only 2–10% O-GlcNAc modified.
Each Click-iT O-GlcNAc Enzymatic Labeling System provides:
We offer several azide-modified isoprenoids and fatty acids, including:
These azide-functionalized isoprenoids and fatty acids enable detection of post-translational lipidation of proteins by in-gel fluorescence scanning, fluorescence microscopy and flow cytometry.
We offer a rich selection of azide- and alkyne-derivatized fluorescent dyes for coupling to complementary azide- and alkyne-functionalized biomolecules (Click Chemistry—Section 3.1, Molecular Probes azide and alkyne derivatives—Table 3.1), including:
We also offer a set of Alexa Fluor, tetramethylrhodamine and biotin DIBO alkyne derivatives for copper-free click reactions with azide-modified targets:
Antibodies to Oregon Green 488, tetramethylrhodamine and Alexa Fluor 488 dyes (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) and Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) are available to provide signal amplification if necessary. The biotin azide and alkyne reagents facilitate western blotting applications and streptavidin enrichment in combination with our streptavidin and CaptAvidin agarose affinity matrices (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6).
In addition to azide- and alkyne-derivatized dyes and biotinylation reagents, we offer three Click-iT Protein Analysis Detection Kits (C33370, C33371, C33372) that provide labeled alkynes for the detection of azide-labeled biomolecules. These Click-iT Protein Analysis Detection Kits provide sufficient reagents for 10 labeling reactions based on the provided protocol and include:
For added convenience, we offer Click-iT Reaction Buffer Kits for protein or cell samples labeled with an azide- or alkyne-tagged biomolecule. The Click-iT Cell Reaction Buffer Kit (C10269) includes sufficient reagents to perform 50 reactions based on a 0.5 mL reaction volume for subsequent analyses by flow cytometry, fluorescence microscopy or high-content screening (HCS). The Click-iT Protein Reaction Buffer Kit (C10276) includes everything required for click coupling to functionalized proteins for subsequent standard protein biochemical analyses (e.g., western blots or mass spectrometry).
We have developed a suite of compatible methodologies for the differential staining of specific proteins (phosphoproteins, glycoproteins or membrane proteins) and the total-protein profile in two or more visually distinguishable colors, producing a more complete picture of the proteome. This set of protein stains not only offer the capacity for the simultaneous detection of multiple protein targets in a single sample, but also provides a combination of high sensitivity and simplicity that can streamline protocols in 1D and 2D polyacrylamide gels or on blots.
The Pro-Q Diamond phosphoprotein gel stain, Pro-Q Emerald glycoprotein gel stains and SYPRO Ruby protein gel stain—which we have optimized to complement each other in selectivity, sensitivity and staining protocols—can be used in serial detection of phosphoproteins, glycoproteins and total proteins on a single protein sample separated by 1D or 2D gel electrophoresis (Figure 9.4.3). Our Rhinohide polyacrylamide gel strengthener (R33400, Protein Detection on Gels, Blots and Arrays—Section 9.3) greatly improves the strength of any polyacrylamide gel, making it easy to perform these multiple staining procedures without special gel handling. After each staining step, an image of the gel is collected. Once collected, the three images can be overlaid in any combination for analysis of phosphorylation, glycosylation and total-protein expression. Because all three stains are used on the same gel, unambiguous spot matching of phosphoproteins and glycoproteins is made simple by direct comparison with the total-protein profile provided by the SYPRO Ruby protein gel stain. This simultaneous measurement of several variables ensures perfect spatial registration of signals and increases the amount of data that can be collected in a single experiment, leading to more controlled experiments, more accurate data comparisons and fewer ambiguities.
Pro-Q Diamond phosphoprotein gel stain is a breakthrough technology that provides a simple, direct method for selectively staining O-linked phosphoproteins in polyacrylamide gels (Figure 9.4.4). It is ideal for the identification of kinase targets in signal transduction pathways and for phosphoproteomic studies. This proprietary fluorescent stain allows direct, in-gel detection of phosphate groups attached to tyrosine, serine or threonine residues. The Pro-Q Diamond phosphoprotein gel stain can be used with standard SDS-polyacrylamide gels (Figure 9.4.4) or with 2D gels (). The simple and reliable staining protocol delivers results in as little as 4 to 5 hours. The stain is also compatible with mass spectrometry, allowing analysis of the phosphorylation state of entire proteomes. The Pro-Q Diamond phosphoprotein gel stain provides:
The Pro-Q Diamond phosphoprotein gel stain (Pro-Q Diamond gel stain reagents and kits—Table 9.4) is supplied ready-to-use in three different sizes: a 200 mL size (P33301) suitable for staining approximately four minigels; a 1 L size (P33300) suitable for staining approximately 20 minigels or two large-format gels, e.g., 2D gels; and a 5 L bulk-packaging size (P33302). In addition, we offer Pro-Q Diamond Phosphoprotein Gel Staining Kits (MPP33300, MPP33301, MPP33302) that include both the Pro-Q Diamond gel stain and the PeppermintStick phosphoprotein molecular weight standards (see below). All products are accompanied by a simple and reliable staining and destaining protocol that delivers results in as little as four to five hours. For convenient destaining, we also offer the Pro-Q Diamond phosphoprotein gel destaining solution as a ready-to-use solution in either a 1 L (P33310) or 5 L (P33311) size.
When used together, the Pro-Q Diamond phosphoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Protein Detection on Gels, Blots and Arrays—Section 9.3) make a powerful combination for proteome analysis. The SYPRO Ruby dye is a total-protein stain that, like the Pro-Q Diamond gel stain, is quantitative over three orders of magnitude. Determining the ratio of the Pro-Q Diamond dye to SYPRO Ruby dye signal intensities for each band or spot thus provides a measure of the phosphorylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Kit #2 with 200 mL of the Pro-Q Diamond phosphoprotein gel stain and 200 mL of the SYPRO Ruby protein gel stain (M33306), the Multiplexed Proteomics Kit #1 with 1 L of each stain (M33305) and the Multiplexed Proteomics Phosphoprotein Gel Stain Kits (MPM33305, MPM33306), which include the Pro-Q Diamond phosphoprotein gel stain, SYPRO Ruby protein gel stain and PeppermintStick phosphoprotein molecular weight standards (see below); Pro-Q Diamond gel stain reagents and kits—Table 9.4 summarizes all of our Pro-Q Diamond gel stain reagents and kits.
The Pro-Q Diamond Phosphoprotein Blot Stain Kit (P33356) provides a simple and quick method for directly detecting phosphoproteins on poly(vinylidene difluoride) (PVDF) or nitrocellulose membranes without the use of radioactivity or antibodies. As with the gel stain, the Pro-Q Diamond phosphoprotein blot stain detects phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins, independent of the sequence context of the phosphorylated amino acid residue. Thus, the native phosphorylation levels of proteins from a variety of sources, including tissue specimens and body fluids, can be analyzed. Protein samples are separated by 1D or 2D gel electrophoresis, electroblotted to the membrane, stained and destained using a protocol similar to that typically performed with amido black or Ponceau S staining of total-protein profiles on membranes. After staining, gels are simply imaged using any of a variety of laser scanners, xenon-arc lamp–based scanners or CCD-based imaging devices employing UV transilluminators; the excitation/emission maxima of the Pro-Q Diamond phosphoprotein blot stain are ~555/580 nm (Figure 9.4.6). The limits of detection for the stain on PVDF membrane blots are typically 8–16 ng of phosphoprotein, with a linear dynamic range of approximately 15-fold. The sensitivity of the Pro-Q Diamond phosphoprotein blot stain is decreased when using nitrocellulose blots. Each Pro-Q Diamond Phosphoprotein Blot Stain Kit provides sufficient reagents for staining ~20 minigel electroblots, including:
The Pro-Q Diamond phosphoprotein blot stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Furthermore, the Pro-Q Diamond phosphoprotein blot stain is compatible with the standard colorimetric, fluorometric and chemiluminescent detection techniques employed in immunoblotting. This phosphoprotein blot stain may be used in conjunction with the SYPRO Ruby protein blot stain, a total-protein stain that is quantitative over two orders of magnitude on blots. Using the SYPRO and Pro-Q Diamond blot stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein.
PeppermintStick phosphoprotein molecular weight standards are a mixture of phosphorylated and nonphosphorylated proteins with molecular weights from 14,400 to 116,250 daltons. Separation by polyacrylamide gel electrophoresis resolves this mixture into two phosphorylated and four nonphosphorylated protein bands (). These standards serve both as molecular weight markers and as positive and negative controls for our Pro-Q Diamond phosphoprotein gel stain and other methods that detect phosphorylated proteins. We offer two different unit sizes of the PeppermintStick phosphoprotein molecular weight standards: a 40 µL unit size sufficient for 20–40 gel lanes (P27167) and a 400 µL unit size sufficient for 200–400 gel lanes (P33350).
Formulated especially for MALDI-MS, the phosphopeptide standard mixture (P33357) contains equimolar amounts of three unphosphorylated and four phosphorylated peptides, ranging in mass between 1047 and 2192 and representing phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) monophosphopeptides, as well as a peptide containing both pT and pY. This mixture is ideal for use as an internal or external control for LC/MS, MALDI analysis or β-elimination reactions.
The Pro-Q Diamond Phosphoprotein Enrichment Kit (P33358) enables efficient, nonradioactive isolation of phosphoproteins from complex cellular extracts. This kit provides resin, reagents and columns designed to isolate phosphoproteins from 0.5–1.0 mg of total cellular protein per column. The column bed volume can be easily scaled up or down depending on the amount of available starting material. The phosphoprotein-binding properties of the resin allow efficient capture of both native and denatured proteins. Therefore, cell or tissue samples can be denatured in lysis buffers and stored in the freezer prior to the phosphoprotein enrichment procedure. Each Pro-Q Diamond Phosphoprotein Enrichment Kit contains:
Phosphoprotein Enrichment Module
Protease Inhibitor and Endonuclease Module
Protocols for both undenatured and denatured lysates are provided, and these procedures can be completed in approximately three hours. For added convenience, the Pro-Q Diamond Phosphoprotein Enrichment and Detection Kit (P33359) provides all the reagents in the Pro-Q Diamond Phosphoprotein Enrichment Kit, as well as Pro-Q Diamond phosphoprotein gel stain and PeppermintStick phosphoprotein molecular weight markers for detecting phosphoproteins on SDS-polyacrylamide gels.
The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) provides a method for selective staining of phosphoproteins or phosphopeptides on microarrays, without the use of antibodies or radioactivity. This kit permits direct detection of phosphate groups attached to tyrosine, serine or threonine residues in a microarray environment and has been optimized for microarrays with acrylamide gel surfaces. Each Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit provides:
The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit is useful for identifying kinase targets in signal transduction pathways and for phosphoproteomics studies.
The Pro-Q Diamond LC Phosphopeptide Detection Kit (P33203) provides sensitive and selective fluorescence-based detection of phosphorylated peptides during liquid chromatography separations. The Pro-Q Diamond LC phosphopeptide detection reagent interacts selectively with phosphoserine-, phosphothreonine- and phosphotyrosine-containing peptides to form highly fluorescent dye–phosphopeptide complexes that elute from an HPLC column with altered retention times, allowing identification and purification of phosphopeptides prior to analysis by mass spectrometry. This kit is ideal for isolating phosphopeptides from chromatographic fractions or from complex peptide mixtures such as the tryptic digest of a phosphoprotein. The Pro-Q Diamond LC Phosphopeptide Detection Kit provides:
Sufficient reagents are provided for 20 HPLC separations; a single separation will selectively detect 20 picomoles or less of a monophosphorylated peptide using a standard microbore C18 HPLC column.
The Antibody Beacon Tyrosine Kinase Assay Kit (A35725), described in detail in Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3, provides a simple yet robust solution assay for real-time monitoring of tyrosine kinase activity and the effectiveness of potential inhibitors and modulators. The key to this tyrosine kinase assay is a small-molecule tracer ligand labeled with our bright green-fluorescent Oregon Green 488 dye. When an anti-phosphotyrosine antibody binds this tracer ligand to form the Antibody Beacon detection complex, the fluorescence of the Oregon Green 488 dye is efficiently quenched. In the presence of a phosphotyrosine-containing peptide, however, this Antibody Beacon detection complex is rapidly disrupted, releasing the tracer ligand and relieving its antibody-induced quenching. Upon its displacement by a phosphotyrosine residue, the Oregon Green 488 dye–labeled tracer ligand exhibits an approximately fourfold enhancement in its fluorescence, enabling the detection of as little as 50 nM phosphotyrosine-containing peptide with excellent signal-to-background discrimination.
Glycoproteins play important roles as cell-surface markers, as well as in cell adhesion, immune recognition and inflammation reactions. To facilitate research on glycoproteins, we offer the Pro-Q Glycoprotein Stain Kits for Gels and for Blots, which provide extraordinary sensitivity, linearity and ease of use for selective detection of glycoproteins.
Pro-Q Emerald 300 and Pro-Q Emerald 488 Glycoprotein Stain Kits (Pro-Q Emerald glycoprotein stain kits for gels and for blots—Table 9.5) provide advanced reagents for detecting glycoproteins in gels and on blots. The Pro-Q Emerald glycoprotein stains react with periodate-oxidized carbohydrate groups, creating a bright green-fluorescent signal on glycoproteins (Figure 9.4.7). The staining procedure requires only three steps: fixation, oxidation and staining—no reduction step is required. Depending on the nature and degree of glycosylation, the Pro-Q Emerald 300 stain allows the detection of as little as 1 ng of a glycoprotein per band in gels (4 ng/band with the Pro-Q Emerald 488 stain), making these stains about 50 times more sensitive than the standard periodic acid–Schiff base method using acidic fuchsin dye. Blot staining is not quite as sensitive (2–18 ng of a glycoprotein per band can be detected) and is more time consuming, but provides an opportunity to combine glycoprotein staining with immunostaining or other blot-based detection techniques. The Pro-Q Emerald 300 stain is best visualized using 300 nm UV illumination, whereas the Pro-Q Emerald 488 stain is best visualized using visible light with wavelengths near its 510 nm excitation maximum. The Pro-Q Emerald dye is also used as the detection reagent in our Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495), which is described in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3.
Figure 9.4.7 Detecting glycoproteins with Pro-Q Emerald glycoprotein detection reagents. Oxidation with periodic acid converts cis-glycols to dialdehydes, which can then react with the hydrazide-based Pro-Q Emerald reagents to form a covalent bond.
The Pro-Q Emerald glycoprotein stains can be combined with general protein stains for dichromatic detection of glycoproteins and total proteins in gels and on blots, making it much easier to identify the location of the glycoproteins in the total-protein profile (Figure 9.4.8, Figure 9.4.9, Figure 9.4.10, ). The SYPRO Ruby protein gel and blot stains (described in Protein Detection on Gels, Blots and Arrays—Section 9.3) provide the same sensitivity as silver staining (gels) or colloidal gold staining (blots) but, unlike these chromogenic techniques, do not require formaldehyde or glutaraldehyde, which can produce false-positive responses when glycoproteins are stained. These total-protein stains make it possible to visualize the entire protein complement of a sample and to thus identify contaminating proteins, to compare stained proteins to molecular weight standards and to provide a control for protease contamination in glycosidase mobility-shift experiments. The SYPRO Ruby protein blot stain is additionally useful for assessing the efficiency of protein transfer to a blot (Figure 9.4.10), which is especially important when working with glycoproteins, because they often transfer poorly to blotting membranes. Proteins labeled with the SYPRO Ruby total-protein stains exhibit red-orange fluorescence when excited with either a 300 nm UV light source or a laser scanner with a 473, 488 or 532 nm laser light source.
The Pro-Q Emerald Glycoprotein Stain Kits also include our CandyCane molecular weight standards, a mixture of glycosylated and nonglycosylated proteins that, when separated by electrophoresis, provide alternating positive and negative controls (Figure 9.4.11). The CandyCane molecular weight standards are also available separately (C21852, see below). In addition, we offer a Pro-Q Emerald 300 Glycoprotein Gel Stain Kit (P21855) that includes our SYPRO Ruby protein gel stain for detecting total proteins. The Pro-Q Emerald Glycoprotein Stain Kits for gels and blots (P21855, P21857, P21875) contain sufficient materials to stain approximately ten 8 cm × 10 cm gels or blots, including:
Figure 9.4.9 Mobility-shift gel assay using deglycosylating enzymes, stained with the SYPRO Ruby protein gel stain (top; S12000, S12001, S21900) and Pro-Q Emerald 300 glycoprotein stain (bottom). The glycoproteins α1-acidic glycoprotein, fetuin and horseradish peroxidase (HRP) are shown before (lanes 2, 4 and 6, respectively) and after (lanes 3, 5 and 7, respectively) glycosidase treatment. Glycosidase treatment resulted in a mobility shift and loss of green-fluorescent Pro-Q Emerald 300 staining for α1-acidic glycoprotein and fetuin, indicating that the carbohydrate groups had been cleaved off. HRP, which contains an α-(1,3)-fucosylated asparagine GlcNac-linkage that is resistant to many glycosidases, showed neither a mobility shift nor a loss of green-fluorescent Pro-Q Emerald 300 staining. Thus, use of the Pro-Q Emerald 300 Glycoprotein Stain Kits (P21855, P21857, M33307) identifies glycoproteins that are not susceptible to the glycosidases used in the assay, providing important structural information about the glycoprotein's carbohydrate moiety.
When used together, the Pro-Q Emerald 300 glycoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Protein Detection on Gels, Blots and Arrays—Section 9.3) make a powerful combination for proteome analysis (Figure 9.4.8). Determining the ratio of the Pro-Q Emerald dye to SYPRO Ruby dye signal intensities for each band or spot provides a measure of the glycosylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a lightly glycosylated, high-abundance protein from a heavily glycosylated, low-abundance protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Glycoprotein Gel Stain Kit with 1 L of our Pro-Q Emerald 300 glycoprotein gel stain and 1 L of our SYPRO Ruby protein gel stain (M33307).
CandyCane glycoprotein molecular weight standards (C21852) contain a mixture of glycosylated and nonglycosylated proteins with molecular weights from 14,000 to 180,000 daltons). When separated by polyacrylamide gel electrophoresis, the standards appear as alternating bands corresponding to glycosylated and nonglycosylated proteins (Figure 9.4.11). Thus, these standards serve both as molecular weight markers and as positive and negative controls for methods that detect glycosylated proteins, such as those provided in our Pro-Q Emerald Glycoprotein Stain Kits (see above).
Figure 9.4.11 Glycosylated and nonglycosylated proteins in the CandyCane glycoprotein molecular weight standards (C21852). The standards were electrophoresed through two identical 13% polyacrylamide gels. Both lanes contain ~0.5 µg of protein in each band. The left lane was stained with our SYPRO Ruby protein gel stain (S12000, S12001, S21900) to detect all eight marker proteins. The right lane was stained using the reagents in the Pro-Q Emerald 300 Glycoprotein Gel Stain Kit (P21855).
The luminescent lanthanide terbium, which is available as its chloride salt (Tb3+ from TbCl3, T1247), selectively stains calcium-binding proteins in SDS-polyacrylamide gels. With some modifications to the staining protocol, these lanthanides can also be used to detect all protein bands. Terbium chloride has also been used as a rapid negative stain for proteins in SDS-polyacrylamide gels, in which the background is green fluorescent and the proteins are unstained.
BOCILLIN FL penicillin and BOCILLIN 650/665 penicillin (B13233, B13234) are green- and infrared-fluorescent penicillin analogs, respectively, that bind selectively and with high affinity to penicillin-binding proteins present on the cytoplasmic membranes of eubacteria. When electrophoresed under nonreducing conditions, the dye-labeled penicillin-binding proteins are easily visible in the gel with sensitivity in the low nanogram range (Figure 9.4.12). BOCILLIN FL penicillin, synthesized from penicillin V and the BODIPY FL dye (spectrally similar to fluorescein), has been used to determine the penicillin-binding protein profiles of Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumoniae, and these binding profiles are found to be similar to those reported by researchers using radioactively labeled penicillin V. Fluorescently labeled penicillin has also been used for direct labeling and rapid detection of whole E. coli and Bacillus licheniformis and of Enterobacter pneumoniae. The β-lactam sensor-transducer (BlaR), an integral membrane protein from Staphylococcus aureus, covalently and stoichiometrically reacts with β-lactam antibiotics, including BOCILLIN FL penicillin, by acylation of its active-site serine residue.
TC-FlAsH and TC-ReAsH detection technology, based on the tetracysteine tag first described by Griffin, Adams and Tsien in 1998, takes advantage of the high-affinity interaction of a biarsenical ligand (FlAsH-EDT2 or ReAsH-EDT2) with the thiols in a tetracysteine (TC) expression tag fused to the protein of interest. The FlAsH-EDT2 ligand is essentially fluorescein that has been modified to contain two arsenic atoms at a set distance from each other, whereas the ReAsH-EDT2 ligand is a similarly modified resorufin. Virtually nonfluorescent in the ethanedithiol (EDT)-bound state, these reagents become highly fluorescent when bound to the thiol-containing tetracysteine tag Cys-Cys-Xxx-Yyy-Cys-Cys, where Xxx-Yyy is typically Pro-Gly (Figure 9.4.13). Modified tags with additional flanking sequences produce higher affinity binding of the biarsenical ligand, resulting in improved signal-to-background characteristics. Selective labeling of two proteins for fluorescence microscopy colocalization and FRET analysis has been accomplished using TC tags with different binding affinities in combination with FlAsH-EDT2 and ReAsH-EDT2.
Transfecting the host cell line with an expression construct comprising the protein of interest fused to a tetracysteine tag (CCPGCC) is the first step in TC-FlAsH TC-ReAsH detection. The tagged protein is then detected by the addition of FlAsH-EDT2 reagent (Figure 9.4.13) or ReAsH-EDT2 reagent, generating green or red fluorescence, respectively, upon binding the tetracysteine motif. For detection of tetracysteine-tagged proteins expressed in cells, we offer the TC-FlAsH II and TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits (T34561, T34562, T34563), which are described in Thiol-Reactive Probes Excited with Visible Light—Section 2.2.
As an alternative to in-cell detection, we offer the TC-FlAsH Expression Analysis Detection Kits (A10067, A10068) for detecting tetracysteine-tagged proteins in polyacrylamide gels (Figure 9.4.14). These kits provide:
Sufficient reagents are provided for ten 17-well minigels, based on a 12 µL reaction volume. When bound to TC-tagged protein, FlAsH dye exhibits excitation/emission maxima of 505/530 nm. The Orange and Red total-protein gel stains supplied in these detection kits exhibit emission maxima of 585/620 nm and 650/660 nm, respectively.
The oligohistidine domain is a Ni2+-binding peptide sequence comprising a string of four to six histidine residues. When the DNA sequence corresponding to the oligohistidine domain is fused in frame with a gene of interest, the resulting recombinant protein can be easily purified using a nickel-chelating resin.
Developed by QIAGEN, the Penta·His mouse IgG1 monoclonal antibody (P21315) provides a sensitive method for specific detection of fusion proteins that have an oligohistidine domain comprising five or six consecutive histidine residues. The antibody does not recognize tetrahistidine domains or domains in which the histidine string is interrupted by another amino acid. The Penta·His antibody binds to the oligohistidine domain regardless of the surrounding amino acid context and even when the group is partially hidden, although subtle differences in the amino acid context may change the sensitivity limit for a particular fusion protein. The antibody is ideal for detecting oligohistidine fusion proteins on western blots (). The Penta·His antibody is also useful for immunoprecipitation, ELISA assays and immunohistochemistry.
Oxidative injury can be monitored by following the formation of protein-derived aldehydes and ketones. Traditionally, protein-derived aldehydes and ketones have been quantitated using a colorimetric assay based on their reaction with 2,4-dinitrophenylhydrazine to yield protein-bound dinitrophenyl (DNP) moieties. A much more sensitive ELISA method has been developed that detects the protein-bound DNP using unlabeled or biotin-labeled anti-DNP antibodies (A6430, A6435; Anti-Dye and Anti-Hapten Antibodies—Section 7.4). The bound anti-DNP antibody is subsequently detected with horseradish peroxidase–conjugated secondary detection reagents (Secondary Immunoreagents—Section 7.2). Our Alexa Fluor 488 and fluorescein conjugates of the anti-DNP antibody (A11097, A6423; Secondary Immunoreagents—Section 7.2) can also be applied to this detection scheme. Our polyclonal antibody to nitrotyrosine (A21285, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) can be used similarly to separate and detect proteins of cell extracts that have been naturally nitrated by nitric oxide (Probes for Nitric Oxide Research—Section 18.3, Figure 9.4.15).
Figure 9.4.15 Specificity of our rabbit anti-nitrotyrosine antibody (A21285) to nitrated proteins. Equal amounts of avidin (A887, lane 1) and CaptAvidin biotin-binding protein (C21385, lane 2) were run on an SDS-polyacrylamide gel (4–20%) and blotted onto a PVDF membrane. CaptAvidin biotin-binding protein, a derivative of avidin, has nitrated tyrosine residues in the biotin-binding site. Nitrated proteins were identified with the anti-nitrotyrosine antibody in combination with an alkaline phosphatase conjugate of goat anti–rabbit IgG antibody (G21079) and the red-fluorescent substrate, DDAO phosphate (D6487).
Biotinylated glutathione ethyl ester (BioGEE, G36000) is a cell-permeant, biotinylated glutathione analog for detecting glutathiolation. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated with BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins. Once these cells are fixed and permeabilized, glutathiolation levels can be detected with a fluorescent streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9) using either flow cytometry or fluorescence microscopy. Proteins glutathiolated with BioGEE can be captured using streptavidin or CaptAvidin agarose (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) and analyzed by mass spectrometry or by western blotting methods.
In protein fusion techniques, the coding sequence of one protein is fused in-frame with another so that the expressed hybrid protein possesses desirable properties of both parent proteins. One common partner in these engineered products is glutathione S-transferase (GST), a protein with natural binding specificity that can be exploited to facilitate its purification. Because the GST portion of the fusion protein retains its affinity and selectivity for glutathione, the fusion protein can be conveniently purified from the cell lysate in a single step by affinity chromatography on glutathione agarose (Figure 9.4.16). For purification of GST fusion proteins, we offer glutathione linked via the sulfur atom to crosslinked beaded agarose (10 mL of sedimented bead suspension, G2879). Each milliliter of gel can bind approximately 5–6 mg of bovine-liver GST. Adding excess free glutathione liberates the GST fragment from the matrix, which can then be regenerated by washing with a high-salt buffer.
We also offer a highly purified rabbit polyclonal anti-GST antibody (A5800) that can be used to purify GST fusion proteins by immunoprecipitation. This highly specific antibody, which was generated against a 260–amino acid N-terminal fragment of the Schistosoma japonica enzyme expressed in Escherichia coli, is also useful for detecting GST fusion proteins on western blots and for detecting GST distribution in cells. The intensely green-fluorescent Alexa Fluor 488 conjugate of anti–glutathione S-transferase (A11131) is also available for direct detection of GST fusion proteins.
Following purification, the fusion protein can serve as an immunogen for antibody production or its properties can be compared with those of the native polypeptide to provide insights on the normal function of the polypeptide of interest. Such methods have been used to investigate biological properties of many proteins. Examples include cleavage of the capsid assembly protein ICP35 by the herpes simplex virus type 1 protease, the role of the Rho GTP-binding protein in lbc oncogene function and the association of v-Src with cortactin in Rous sarcoma virus–transformed cells. In fact, the Ca2+-binding properties of a protein kinase C–GST fusion protein were examined while the GST fusion protein was still bound to the glutathione agarose. Likewise, interactions of a DNA-binding protein–GST fusion protein have been assessed using an affinity column consisting of the fusion protein bound to glutathione agarose. Alternatively, the GST fusion expression vector can be engineered to encode a recognition sequence for a site-specific protease, such as thrombin or factor Xa, between the GST structural gene and gene of interest. Once the fusion protein is bound to the affinity matrix, the site-specific enzyme can be added to release the protein.
Figure 9.4.16 Coomassie brilliant blue–stained SDS-polyacrylamide gel, demonstrating the purification of a glutathione S-transferase (GST) fusion protein using glutathione agarose (G2879). Lane 1 contains crude supernatant from an Escherichia coli lysate and lane 2 contains the affinity-purified GST fusion protein.
Streptavidin acrylamide (S21379), which is prepared from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid (acryloyl-X, SE, A20770), may be useful for the preparation of biosensors. A similar streptavidin acrylamide has been shown to copolymerize with acrylamide on a polymeric surface to create a uniform monolayer of the immobilized protein. The streptavidin can then bind biotinylated ligands, including biotinylated hybridization probes, enzymes, antibodies and drugs.
Like streptavidin and CaptAvidin biotin-binding protein, other amine-containing biomolecules can be crosslinked to acrylamides using acryloyl-X, SE. Acryloyl-X, SE reacts with amines of proteins, amine-modified nucleic acids and other biomolecules to yield acrylamides that can be copolymerized into polyacrylamide matrices or on surfaces, such as in microarrays and in biosensors. We prepare both streptavidin and CaptAvidin biotin-binding protein conjugated to 4% beaded crosslinked agarose (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6)—matrices that can be used to isolate biotinylated peptides, proteins, hybridization probes, haptens and other molecules.
For Research Use Only. Not for use in diagnostic procedures.


text:
Parshat Beshalach
Pharaoh finally sends B'nei Yisrael out of Egypt. With pillars of cloud and fire, G-d leads them toward Eretz Yisrael on a circuitous route, avoiding the Pelishtim (Philistines). Pharaoh regrets the loss of so many slaves and chases the Jews with his army. The Jews are very afraid as the Egyptians draw close, but G-d protects them. Moshe raises his staff and G-d splits the sea, enabling the Jews to cross safely. Pharaoh, his heart hardened by G-d, commands his army to pursue, whereupon the waters crash down upon the Egyptian army. Moshe and Miriam lead the men and women, respectively, in a song of thanks. After three days' travel only to find bitter waters at Marah, the people complain. Moshe miraculously produces potable water. In Marah they receive certain mitzvot. The people complain that they ate better food in Egypt. G-d sends quail for meat and provides manna, a miraculous bread that falls from the sky every day except Shabbat. On Friday a double portion descends to supply the Shabbat needs. No one is able to obtain more than his daily portion, but manna collected on Friday suffices for two days so the Jews can rest on Shabbat. Some manna is set aside as a memorial for future generations. When the Jews again complain about a lack of water, Moshe miraculously produces water from a rock. Then Amalek attacks. Joshua leads the Jews in battle while Moshe prays for their welfare.
The Essence Of Love
"G-d said to Moshe, Why do you cry out to me..? "(14:15)
No one knew G-d like Moshe. No one spoke to Him "face-to-Face" like Moshe. Why, then, did Moshe cry out to G-d when the Jewish People started to panic at the sight of Pharaoh and his hordes bearing down on them? Surely he, of all people, was confident that G-d would rescue them.
Empathy is the essence of love. Whats important to you is important to me. What concerns you, concerns me. Moshe cried out to G-d not because he had any doubt that G-d would save them, but because he loved the Jewish People so much that he couldnt bear to see them gripped by mortal fear.
- Based on the Sfat Emet
Reasons To Be Miserable?
"G-d will fight for you and you will remain silent." (14:14)
Since 2000, anti-Semitic incidents in Germany have increased by 69%. Violent anti-Semitic crimes have jumped up nearly 100%. Similarly, Austrian monitoring agencies report that from 2002 to 2003 there was a 70% increase in anti-Semitic attacks and incidents. Of the 313 racist incidents reported in 2002 in France, 193 were directed at the Jewish community, 6 times more than in 2001.
Up until the year 2002, in Australia the annual average number of anti-Semitic incidents was 279. In 2003 there were 481 reports of anti-Semitic incidents, including a notable increase in physical violence and property damage (up 60% from 2002).
Its easy to become despondent at the state of things. Every year seems to bring yet another downward turn, whether in the growing statistics of world-wide anti-Semitism, or, nearer to home, those who try and demonize Torah Jews with rhetoric that would have made Der Sturmer proud.
We dont seem to have much going for us at the moment.
However, theres one reason that should give us great optimism.
When G-d took us out of Egypt, He was, so to speak, duty-bound to protect us against our enemies. He led us out into a trackless wilderness. At that time, as it were, He was "obliged" to hear our pleas.
"G-d will fight for you and you will remain silent."
Its not just when we have the right to petition that G-d will fight our enemies. Even when we have nothing to claim in our defense, when the Jewish People are very far from being able to maintain that they should be saved; when all we can do is to "remain silent" with nothing to say in our defense, even then will G-d save us from our enemies.
- Based on The Meshech Chochma

subset: commoncrawl


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How well do you describe what your company does? There are few sales tools more valuable than a concise, interesting "pitch" that gets listeners as thrilled about your company as you are. It's something you need to use when you're making a sales presentation, seeking financing, networking at an event, or even just chatting with a neighbor.
Creating a great pitch is not as easy as it sounds. And even if you have one that you think works, it may be in need of fine-tuning. Often, you're too close to the details of your company to remember what will interest others. Or you've described your company so many times that you start to sound robotic. Successful pitching often comes down to a matter of content and style. First, you need to pare down what you do to a few succinct sentences that someone unfamiliar with your business or even your industry can quickly grasp. Those words then need to be delivered with passion and verve that convey your excitement.
Follow these guidelines to develop or refine a pitch that sizzles:
Open with what makes you unique
Start your pitch with what sets your business apart – your unique selling proposition or USP. You need to break the ice quickly, so avoid describing yourself in run-of-the-mill terms. "I run a food store" is not nearly as compelling as "I sell gourmet foods from local artisans and regional, organic farms." Some people start out with a tagline – a memorable phrase or wordplay that focuses interest and quickly encapsulates their company's vibe. For example, a company that database segmentation and prospecting for clients might say, "We mine business data to find the gold."
Focus on problems and solutions
Every successful product or service solves some kind of problem. Consider the market need that your company fills. A successful pitch conveys this as a story with a happy ending – the problem your target market faces, and how your company's approach alleviates that pain. Clearly lay out the benefits you provide – remember, it's not how a product or service works, but what it does for the person who will buy it. Convey the ways you help customers save time, save money, be more productive or lead easier lives.
Provide proof
Interweave relevant facts into your pitch to support your claims, prove your benefits and show your success. Tell your prospect how much repeat business you generate or how much you save a customer who signs up for your service. When appropriate, cite sales numbers, distribution and customer relationships or statistics that demonstrate market potential. If you've been recognized with awards or other honors, mention them to back up your claims. Different Pitches for Different Audiences There are number of situations where you'll need to describe your company. Here's how to tailor your pitch to each audience.
Be tangible
Don't get caught up in jargon, acronyms, clichés or meaningless terms. Avoid sounding like a technology manual or a self-help guide – this can make you seem dull, boring and out of touch. Instead, express your benefits in a way that can be understood by the "man on the street."
Show your passion
The perfect pitch appeals to the emotions, not the intellect. You need to connect on a very real level with your audience. Use emotion-laden terms to describe why your business makes a difference. Think back, as well, to what got you excited about starting your business in the first place – that story can often communicate the passion and delight you feel about your company.
Practice, practice, practice
Spend enough time practicing your pitch so it sounds unrehearsed. That way, when you use it, your delivery will seem effortless and natural. Go through your pitch with someone who doesn't know much about your business – a friend or a colleague in another industry, for instance – to get some unbiased input. You'll find that pitching gets easier with time. And, if you think of yourself as a storyteller – who just happens to be telling a business story – you can relax and enjoy yourself.
Finding and Sharing Your Passion
When describing your business to someone new, you want to be more like a proud parent showing off new baby pictures than a pitchman pushing product. It's your passion, enthusiasm and connection that make a pitch memorable.
How do you keep the energy after the 200th repetition?
• Make it personal. Explain what motivated you to start your business or describe what you love about what you do. Discuss how your customers or clients have benefitted.
• Show feeling. When relating your message, use emotion-packed words to generate excitement and passion. My customers help build our business by referring new business to us. I'm so grateful for their faith in us.
• Tell a story. Everyone loves a tale, and it makes it easier to remember you and your key points. Relate an anecdote about a company with a similar problem to theirs and how your solution helped them.
• Keep it current. Try weaving in a newsworthy item or an emerging business trend appropriate to your audience.
• Provide backup. Cite meaningful statistics to reinforce your message. We sold three times as many widgets last quarter as we did a year ago. Whatever you do, let your personality shine through. And remember, no one knows your business the way you do. So go out and tell your story.
Use this worksheet to develop a concise, winning pitch for your business.
• Personalize the information to your audience
• Avoid jargon and clichés
• Develop a flow that makes the biggest impact
• Say it in a way that's comfortable for you
1. What do you do? Explain what your company does:
2. What makes you unique? List some ways you are different from your competition:
3. Who is your product/service for? Review the characteristics of your target audience:
4. What need do you fill? Describe the problems your target market faces and how your product/service solves it:
5. How do you earn money? Provide an overview of your revenue model:
6. Who is on your team? Discuss the unique experience of members of your organization:
7. How have you helped a customer? Illustrate your benefits with a specific example of how you've solved a problem:
8. Why did you start your company? Think about the reasons you opened your business in the first place:
9. What gets you excited about your company? Make an inventory of why your business is important to you:
10. What else do you need to do? Focus on the purpose of your pitch: what information do you need to provide to close your pitch and move to the next step:
The information contained in this document is meant for advisory purposes only. American Express accepts no liability for any outcome of its use.
Do you have a question about business pitches? Connect with a mentor online or in your community today!Copyright © 2024 SCORE Association, SCORE.org
Funded, in part, through a Cooperative Agreement with the U.S. Small Business Administration. All opinions, and/or recommendations expressed herein are those of the author(s) and do not necessarily reflect the views of the SBA.


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The anger over the decision not to allow women bishops in the Church of England continues to intensify with scathing criticisms from those within and out of the church including the Archbishop of Canterbury.
But as the controversial decision was picked apart, a small story about the first female African Anglican bishop went mostly unnoticed.
Ellinah Wamukoya, 61, was consecrated on Saturday four days before the CofE voted against allowing women to become bishops. She will be the church's bishop in Swaziland, her native country.
Her ordination comes two weeks after Fauzia Yusuf Haji Adan was chosen to be Somalia's first female foreign minister.
In April, the Guardian's Afua Hirsch wrote a critical piece of the "west's lazy reporting on Africa" decrying the mass reporting of Africa in the mainstream media when war intensified: "Even worse is the situation when an impromptu Africa season is triggered by newsworthy events in Africa. A dramatic climax in a long-running war, preferably with the close involvement of a western power, usually leads to an African country being "discovered" by the international media."
Looking at statistics from the Inter-Parliamentary Union and UN Women from January this year makes for more depressing reading for women in the UK.
According to the IPU, the UK is 48th in the world in terms of the number of female ministers, behind 25 African countries and 53rd, behind 15, when it comes to the figures for the number of female parliamentary represenatives.
Of course the situation in Africa for women is not perfect like in many parts of the world including the UK, but the developments like this sometimes surpass our own – a fact we would do well to take note of.


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Multiband compression has traditionally been associated with the mastering stage of production, FabFilter's Pro-MB plans to shake things up...
Over the last few years, multiband dynamics - particularly multiband compression - has become a standard tool in the modern producer's toolkit. Once you've got to grips with the basics of EQ and compression, the ability to manipulate the dynamics of specific frequency ranges seems like the inevitable next step.
The effects processors required to do it first found their place in the mastering studio, embraced by engineers for their ability to apply effective dynamics control to selected frequency areas of finished stereo mixes.
As a consequence, most multiband compressor plugins are designed with the mastering stage in mind and, due to the multiple stages of processing involved, have tended to be very demanding on CPU resources and/or not as sonically flexible as they could be.
Despite this, many mix engineers and sound designers have found the raw audio sculpting power of the multiband compressor to be invaluable, putting up with its mastering-orientated design and workflow in order to take advantage of it.
Multiband for the masses
Enter the much-anticipated FabFilter Pro-MB (VST/AU/RTAS/AAX), which aims to bring multiband compression out of the mastering studio and into every stage of the production process, without nailing your CPU to the wall, sporting the cleanest, clearest interface we've ever seen in such a plugin. In typical FabFilter style, the "top level" is quite bare, but there are plenty of controls tucked away beneath it, ready to be called up with a click when required.
The first thing you'll notice when you push an audio signal through Pro-MB is the spectrum analyser in the background of the GUI. The speed and resolution of its analysis are adjustable, and we found it to have a significantly faster response than the analyser in FabFilter's Pro-Q, which Pro-MB's interface obviously has a lot in common with.
Pro-MB can utilise up to six bands, and clicking anywhere in the main window creates the first of them, calling up a bank of familiar dynamics controls with which to set it up: threshold, ratio, attack, release, etc.
The frequency range that this first band works on is indicated by its tinted background and dotted- line crossovers, which can be freely dragged left and right. When you click in empty space to create a second band, you'll notice that, unlike conventional multiband compressors, the bands don't have to be adjacent. In other words, they don't share boundaries/crossovers by default, although you can drag them together to merge them, and click the scissors icon that appears on the crossover when moused over to split adjacent ones.
This is a significant innovation as with any other multiband compressor, you'd have to use a third band as a separator if you wanted the other two to work on non- contiguous areas. Or, to put it another way: replicating Pro-MB's six non-contiguous band processing (including adjustable high and low extremes on the outer two) on a typical multiband compressor would require the use of 12 crossovers.
Crossing the line
While dragging a crossover left and right sets its cutoff frequency, dragging up and down on the filter icon in the middle of it sets the cutoff slope anywhere from 6-48dB/ octave. With each band having two borders, this allows them to be bent into asymmetrical shapes. Dragging the Range handle in a band, meanwhile, sets its maximum gain reduction. The whole system is exceptionally intuitive and quick, enhancing the plugin's suitability for mixing and creative purposes as well as mastering jobs.
The sound of Pro-MB is amazingly transparent, especially in the default Dynamic Phase mode. As well as compression, each band can be switched to Expand mode, for adding punch to specific ranges by boosting (or reducing) the signal when it exceeds (or drops below) the threshold. The compression and expansion algorithms seem to have been very well fine-tuned, enabling you to jump straight in and get good results within a minute or so on most sources.
Of course, it's possible to use Pro-MB as a single-band compressor or expander, too, in which case it's comparable but not identical to Pro-C from the same series - Pro-C is the more feature-rich (albeit single- band) plugin, generally, but Pro-MB has a variable knee control while Pro-C only has two fixed knee shapes.
It seems like Pro-MB may well have it all: innovation, ease of use and a surprisingly low impact on the host computer's resources. It also features dithering and oversampling for when used as a mastering tool, as well as the option to specify the sidechain detector frequency range for each band, regardless of the range it actually operates on, helping with intricate mixing tasks.
The geekier producer will absolutely love it, and thanks to its accessible interface and the sheer range of applications it can turn its hand to, we'd wholeheartedly recommend it to anyone after a more sophisticated process for those occasions when regular EQ and/or compression fall short.


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New York: A breakthrough in DNA analysis is helping identify more victims of the September 11, 2001, attacks in New York, but the scientific advance is of little consolation for families of those whose remains may have been buried in a Staten Island landfill.
The official death toll in the attacks on lower Manhattan's World Trade Center is 2,753, including the missing and presumed dead.
Only 1,642 of them, or about 60 per cent, have been positively identified.
The New York City Medical Examiner's Office has worked for 17 years to identify the remaining 1,100 victims.
Using advances in DNA extraction techniques over the past five years, it has made five more identifications.
The advances have been bittersweet for 9/11 families who unsuccessfully fought to stop the city from making a park out of Staten Island's enormous Fresh Kills landfill, where 1.8 million tons of Twin Towers debris was dumped and buried.
"We are grateful that the identification continues, but there is more material that could have been part of that had the city not been so cavalier with us," said Diane Horning, who led a failed court battle by a group called World Trade Center Families for Proper Burial that hoped block the park project.
Horning led the group, although her son Matthew was one of those identified early on.
Matthew, 26, a database administrator for an insurance company, was working the 95th floor of the North Tower when the planes hit.
New York's Second Circuit Court of Appeals found in 2009 that accusations that the city had mishandled the remains at Fresh Kills amounted to "lack of due care," which was not sufficient to successfully sue the city.
New York officials said at the time that the city did not intend to be insensitive or offend victims' families.
To create the park, Fresh Kills Landfill was covered with layers of soil and other materials to prevent the release of toxic gas from decomposing trash into the atmosphere, according to the Freshkills Park Alliance, New York City's nonprofit partner in developing the park.
Charles Wolf lost his wife Katherine on September 11 and her remains have not been identified.
If they are in the sealed landfill, he considers it "God's will" and he is "at peace" with it.
"What's the remedy? Dig everything up and risk exposing all those toxins again to the environment?, Wolf said.
"No, that's not the answer, because all of a sudden now the cure is worse than the disease."
The ability to identify more victims is the latest chapter in a saga of pain that began on the morning of September 11, 2001, when two airliners crashed into the Twin Towers of the World Trade Center.
Destruction of the Twin Towers was part of the coordinated hijackings of four airliners by Al Qaeda militants that killed nearly 3,000 people in New York, Washington and western Pennsylvania, where one of the planes crashed in a farm field.
The attacks triggered an escalation of US military involvement in the Middle East that persists to this day.
A scientific breakthrough in the extraction of genetic material was made this year and announced by the New York City chief medical examiner last week, as the 17th anniversary of the attacks approached.
The new technique places bone fragments in a chamber containing liquid nitrogen to make them more fragile so they can be pulverised into fine powder.
The more a bone is pulverized, the more likely it becomes to extract DNA.
It is the latest effort in the largest forensic investigation in US history, involving a medical examiner's team of 10 scientists working on remains once thought too degraded from jet fuel, heat and other conditions to undergo testing.
"We're going back to the same remains that we've tried five, 10, 15 times," Mark Desire, who heads the Medical Examiner's crime lab, said in the briefing last week.
"We are making DNA profiles from remains we had no hopes of identifying in the past," he added.
Wolf, who was not among those who opposed the Freshkills Park project, was gratified by the renewed effort.
"It warms my heart that possibly there will be remains found for people who still want them," he said.
"I've gone through a lot of trauma with nothing to grieve over," Wolf said, choking up in a telephone interview.
"I remember watching Nancy Reagan touch her husband's casket. I miss not having that."


text:
There has been some confusion regarding what is actually needed to use the Analysis Services Connector for Power BI. Originally there was a requirement that Azure Active Directory DirSync was required to get this to work. However, this was altered to indicate the following.
Why you might need use DirSync to connect to an on-premises Analysis Services Serverhttps://support.powerbi.com/knowledgebase/articles/505323-why-you-need-dirsync-to-connect-to-on-premises-ana
I wanted to walk through this a little bit and show how this actually works as it may be a little hard to wrap your head around. In this example, I will show you why you would not need DirSync to get this to work.
My tenant has several domains registered with it.
- guyinacube.com
- anothercube.guyinacube.com
- battlestarcloud.com
- guyinacube.onmicrosoft.com
Of these, guyinacube.com and anothercube.guyinacube.com are taking advantage of DirSync. Battlestarcloud.com is not. I want to focus on the battlestarcloud.com domain for this walkthrough. guyinacube.com VMs are local on my workstation. battlestacloud.com is on VM's within Windows Azure. I do have a two way trust between the two forests though. However, the Battlestarcloud.com accounts are not being synced to my O365 Tenant.
If your domain isn't listed, and you want to add it to your O365 Tenant you can look here on how to configure that.
You can see what accounts are synced with Active Directory within the Users list in the Office 365 Admin Portal.
You will notice that for firstname.lastname@example.com it shows In Cloud. I actually created this account within O365 itself, but I do have that account created within the local Active Directory on my VM's. Also, within Azure Active Directory, you can see where the users are from.
The domain, battlestarcloud.com, is still listed within Azure Active Directory though, as it needs to know that the domain is verified.
I just don't have DirSync configured within that domain.
At this point we can configure the Analysis Services Connector on a machine within our battlestarcloud.com domain. For the Account used with the Connector, I used BATTLESTAR\pbiconnector.
I have a Tabular Instance on a machine called CAPTTHRACE and the instance name is TABULAR. Within there I have two Tabular Databases based on AdventureWorks.
After that, I can sign in to PowerBI.com with my email@example.com account. Within Power BI, we can see that the AS Connector is configured on CAPTTHRACE.
When we click on CAPTTHRACE, we can see the list of Models available to use on that server. The fact that we see the list of models indicate that we were able to successfully connect to the Tabular instance with the AS Connector.
We can click on one of the models, and it will then be listed in our Dataset area within the main screen.
When we click on the dataset, it will take us to a blank report canvas. From here we can drag a few fields to create a chart and the data that is used for this is coming from the Tabular Instance on the CAPTTHRACE machine using a credential that we have not used DirSync for.
If we look at a SQL Profiler Trace, against that Tabular Instance, we can see what is actually happening.
The Connector will actually connect with the account you specified during configuration. In this case it is BATTLESTAR\pbiconnector. This account will need Admin rights on the server so that it make use of the EffectiveUserName property. We can see that EffectiveUserName was set with firstname.lastname@example.com. We will use whatever is sent to use from Azure Active Director for the username. Whatever account that is sent by AAD will be used for the EffectiveUserName property. From Analysis Services perspective, this account needs to be a valid account within the local Active Directory. Which in this case it is.
If we try with another user, that doesn't have access to the Tabular Instance, we would see the following:
I was trying to use this with firstname.lastname@example.com which is not listed as an admin and doesn't have rights to the database itself. So, we see nothing come back.
Why do we need DirSync?
We would need it if we had a tenant that only had a default domain. So, something like mytenant.onmicrosoft.com. If we had email@example.com, that is what would be passed for EffectiveUserName. That obviously won't work as that won't be a valid user within your local environment. You could configure DirSync at that point. With DirSync enabled, it will copy your users from your local Active Director to Azure Active Direct and accounts would be created within the O365 Tenant. At that point you can sign in using that credential/email address.
Adam W. Saxton | Microsoft Business Intelligence Support – Escalation Services@GuyInACube | YouTube | Facebook.com\guyinacube


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In his State of the Union address, President Obama announced the goal of cutting energy waste in buildings and homes in half over the next 20 years. House Speaker John Boehner clapped approvingly. U.S. buildings and homes waste so much energy that a 50% reduction of such energy waste would save businesses and individuals billions of dollars, would deliver healthier air to all Americans and would put us on the path of energy independence. Most of our energy comes from burning fossil fuels; so, consuming less fossil fuel will reduce toxic emissions and improve air quality. Cleaner air will save lives. Studies estimate that over 35,000 Americans die every year due to air pollution related illnesses.
Cutting energy waste in half won't just happen on its own, though, and it won't be easy. We need to identify the opportunities where we can eliminate energy waste, and then invest in the types of technologies that lead to more energy efficient buildings and homes. The good news is that these modern, cost-effective technologies are available now.
Clearly, opening windows when a building is overheated is not the solution. For example, building owners will need to invest in control technologies that cut overheating and turn off lights and equipment when not needed. These are smart energy efficiency investments with typically short pay-back periods. And, in reducing the energy we waste, we improve our quality of life with more money in our pockets and fresher air in our lungs.
Finally, let's not forget about the environmental impacts of energy exploration, which is another reason why we shouldn't waste the energy that was so hard to get out of the ground in the first place. The actual extraction of fossil fuels is the second biggest source of U.S. greenhouse gas emissions, and – if developed irresponsibly – can pollute our water, air and oceans — jeopardizing our health, livelihoods and quality of life. When you consider the whole range of health and environmental impacts involved with using, and (of course) wasting, energy –it is blatantly obvious that wasting energy is already coming back to hurt us.
If Washington can agree that wasting energy is senseless, let's keep the momentum going and support smart efforts, policies and investment tools that will help energy efficiency reach its full potential. Cutting energy waste is a win for our wallets, our health and our children's' future.


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As kids clad in their spookiest, scariest get-ups head out to trick-or-treat this Halloween, Ethan Smallwood will be dressing up as a Canadian icon and raising money for a good cause.
Smallwood, 7, from Clarke's Beach, N.L. will be going door-to-door in a Marathon of Hope t-shirt, dressed as his favourite hero Terry Fox.
Instead of collecting candy, Smallwood will be raising money for the Terry Fox Foundation and cancer research. The young boy has already raised more than $13,000 since his mom, Candice Smallwood, posted a video unveiling the special costume on Facebook earlier this month.
This year marks the 38th anniversary of Fox's iconic battle with osteosarcoma, captivating Canadians with his heroism and resilience to raise awareness for cancer research by running across the country.
Fox died one month shy of his 23rd birthday on June 28, 1981.
READ MORE: Canadian icon and hero Terry Fox died 38 years ago today
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Climate Experts INC
Air Conditioning Experts in Melbourne, FL
Climate Experts Inc is a Melbourne-based HVAC company that has won numerous awards. We provide the most current technology, including paperless (Environmentally-friendly) invoicing, online book, on-the-way text, and email notifications. Our goal is to educate our customers in a way that is easy to understand. No hype. No confusing tech talk. No over-promising. No over-promising. Just honest, detailed recommendations. All of our HVAC installations and repairs are guaranteed and warrantied. We encourage you to browse our website, meet the team, read our reviews and see what our happy clients have to say. Call us today for a free estimate on HVAC installation in Melbourne FL. We offer quick online booking and easy financing
Melbourne ( (listen) MEL-bən, also MEL-bərn) is the capital and most-populous city of the Australian state of Victoria, and the second-most populous city in both Australia and Oceania. Its name generally refers to a 9,993 km2 (3,858 sq mi) metropolitan area known as Greater Melbourne, comprising an urban agglomeration of 31 local municipalities, although the name is also used specifically for the local municipality of City of Melbourne based around its central business area. The city occupies much of the northern and eastern coastlines of Port Phillip Bay and spreads into the Mornington Peninsula and the hinterlands towards the Yarra Valley, the Dandenong and Macedon Ranges. It has a population over 5 million (19% of the population of Australia, as per 2020), mostly residing to the east side of the city centre, and its inhabitants are commonly referred to as "Melburnians".Home to Aboriginal peoples for over 40,000 years, the Melbourne area served as a popular meeting place for local Kulin nation clans, Naarm being the traditional Boon wurrung name for Port Phillip Bay. A short-lived penal settlement was built at Port Phillip, then part of the British colony of New South Wales, in 1803, but it was not until 1835, with the arrival of free settlers from Van Diemen's Land (modern-day Tasmania), that Melbourne was founded.
Alamanda Key, Annie Laurie Gardens, Bayberry Estates, Booker Heights, Carriage Park, Clements Woods, Cliftonedge, Coquina Ridge, Crystal Lakes, Daintree


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SAN FRANCISCO — Facebook co-founder and former Mark Zuckerberg roommate Dustin Moskovitz is by many accounts the world's youngest self-made billionaire. But the 27-year-old isn't sipping champagne in the Caribbean.
Instead he's thrown himself back into San Francisco's startup mix, even as Facebook's looming IPO seems likely to send his wealth spiraling even higher.
Moskovitz and his friend Justin Rosenstein, a former Facebooker himself worth $150 million, head a company called Asana, which just launched the first paid version of its online project management service. During a recent interview at their inconspicuous Mission District offices, the pair said they come to work every day because, their fortunes already made, they still have to do something with their lives.
"When we think of work, we think of work as an act of service, as an act of love for humanity," said Rosenstein, 28.
Added Moskovitz: "If we were just retired, we wouldn't be serving anyone."
While such idealistic sentiments might sound too easy coming from two guys who never have to worry about money again, they both do keep working even though they'd never have to again. And like Zuckerberg himself they seem uninterested in the flash and status-hoarding that great wealth makes possible.
In keeping with the recent startup trend of shunning hierarchies, the pair do not have separate offices but sit among the other employees at Asana, which numbers 24 in all. They don't have an entourage. Rosenstein likes to cycle (he recently had his bike stolen).
Also like Zuckerberg, they dress down, Moskovitz in an untucked shirt, Rosenstein in a sweater and Chuck Taylors. On the streets of their neighborhood, which brims with twenty-something hipster geeks, they'd blend right in.
What sets them apart, they acknowledge, is their absolute freedom to pursue their particular vision of how to change the world. And they seem to have no doubt that their software will do just that. After all, as some of Facebook's earliest engineers, they've seen their code change the world once already.
Asana will speed human progress by changing the way people work together, Rosenstein said. Too much time at work is spent doing "work about work," Moskovitz said. They say Asana will free people up to do more important things.
"We could go work on curing cancer. We could go work on building spaceships. We could go work on art projects," Rosenstein said. "What's fun about working at Asana is we get to work on all of them at the same time." Or as Moskovitz, the more circumspect of the two, said, "We're working on a meta-problem."
Whether Asana's world-changing potential exceeds that of competitors in the crowded project and task management software marketplace remains to be seen. Like other similar products, their software lets users set up Web-based to-do lists that any group focused on a common goal can use to assign jobs and keep track of what gets done.
The pair believe Asana will win out on its speed, versatility and ability to maintain the flow alluded to in its name, which in yoga refers to the poses meant to aid the flow of spiritual energy in the body. (Regular yoga sessions are among the perks of office life at Asana. Other benefits include an in-house chef and $10,000 for new hires to set up their desks with whatever computing gear they want.)
Several marquee tech companies have embraced Asana, the company reports, such as Twitter, LinkedIn and Foursquare. Individual backers betting on Asana include venture capital celebrities like Peter Thiel and Mark Andreessen, as well as several of Facebook's earliest employees.
None go as far back as Moskovitz, however. He spent two years at Harvard where he helped Zuckerberg start the site before they dropped out and moved to Palo Alto. He left Facebook in 2008 and started Asana with Rosenstein.
According to Facebook's pre-IPO filing with the Securities and Exchange Commission, Moskovitz holds nearly 134 million shares of Facebook stock, giving him a 7.6 percent stake in the company. Based on the value placed on Facebook's stock in its $1 billion cash-and-stock deal for Instagram, Moskovitz's pre-IPO net worth stands at more than $4 billion.
Like many of the richest Americans, Moskovitz has signed a pledge initiated by Bill and Melinda Gates and Warren Buffett to give away most of his money. His wife runs his philanthropic foundation, which is still getting off the ground.
For now, he said he's focused "110 percent" on Asana. To hear Moskovitz tell it, the choice of coming to the office doesn't come at the expense of some wished-for life of luxury.
"It feels very much like a default. Of course you do that," Moskovitz said. "We're fortunate not to have things that would distract us from being able to act."


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1. Is it a crime to buy and travel on one-way air tickets?
2. Are all relatives of a terrorist considered terrorists?
3. Dr. Haneef's SIM card was not involved in any terror activity.
4. Why was his wife's visa to Australia canceled?
5. When Dr. Haneef's visa was cancelled, why was he not deported?
6. Why was Dr. Asif Ali, friend of Dr. Haneef, harassed and why is he not allowed to say what happened to him when he was under detention?
- Nick Bryant's piece on the Keystone cops - why the case disintegrated.
- Haneef's win new blow for Howard government - court quashes cancellation of visa


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Candy Spelling's Mansion -- CLOSED
Candy Spelling's Mansion CLOSED
It's official ... 22-year-old Petra Ecclestone is now the rightful owner of Candy Spelling's 57,000 square foot mansion in Holmby Hills after the escrow period finally ended today.
Ecclestone (right) -- daughter of Formula 1 racing mogul Bernie Ecclestone -- bought the estate for a cool $85 mil ... a STEAL considering Candy originally wanted $150 mil for the place.
The estate has a bowling alley, beauty salon, several gift-wrapping rooms and parking for 100 cars.
Don't feel too bad for Candy -- she bought 2 penthouse units in a nearby condo complex in Century City for $35 mil ... and plans to move in sometime next year ... after some intense renovations.


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Scary, huh??
One thing that pleasantly surprised me about Astoria was the plethora of butchers and fish shops around the turn of every corner. It's a food wonderland at your disposal. (Not to mention the fruit and vegetable stand right below my apartment. Can you get more convenient?) Just on my walk to the post office the other day, I encountered three fish markets.
One of our first Greek restaurant experiences after the move to Astoria was a restaurant where I was forced to choose between sensible shrimp or the boldness of sardines. I'd always heard fresh sardines were worthy of a try but shrimp was safer. I've regretted passing up the sardines ever since because it was so uncharacteristic of me to make the safe menu selection. How disappointing of me. There's no better way to remedy this than to return home from the fish market with a mass of mussels and sardines for dinner.
There's not much on the Internet in the way of sardine recipes or in any of my cookbooks, except in Mark Bittman's "How to Cook Everything," a book I am generally so-so on. The book informed me that sardines are a popular appetizer throughout Europe and that in the United States, sardines are usually sold "gutted with their heads on." Wrong. Granted, it was my fault for assuming this was so and not even thoroughly inspecting them in the fish market.
Lucky for me (I think...), the book also provided a rough guideline to gutting the things, and Ryan agreed to help. How romantic to gut fish together, considering it was Valentine's Day. So Ryan took my kitchen shears to cut through the belly of the fish and I pulled all the guts out. Despite all my complaining, it wasn't so bad of a task except that it's a tiny bit disgusting, and I was sorry the process had to be repeated 12 times. Why had I gotten so many sardines again?
And it never ended. Each one had to be washed, scaled, stuffed with herbs and slathered with butter. I was getting mighty sick of those sardines and it wasn't even time to eat. Then they pop into the broiler for 3 minutes on each side. After all our hard work, it was time to enjoy those sardines. It's really remarkable how many bones are in them. So much so that one sardine yields maybe two baby bites of unboned fish. But you chew those paranoid still of more bones. Bones, bones, and more bones. We finally just gave up. Ryan thinks lobster isn't worth the effort it yields, which I disagree with. But these really weren't worth the effort.
Fortunately, I had also sauteed fennel and steamed mussels, which were both a smashing success. So two out of three isn't bad. Plus, I could have made a whole meal from the mussels and bread so we didn't even miss the sardines.
The mussels were quick and easy.. And not too bad to clean, considering how much time I put into the sardines. I just soaked the mussels in water for an hour or so and then trimmed off the beard with my kitchen shears (My kitchen shears really got a work out that day.).
The mussels were cooked with some white wine, herbs and a pat of butter in a pan with a lid over them for about seven minutes. The whole process was quick, easy and deliriously delicious. It was the opposite of the sardine fiasco. So skip any fresh sardine fantasy you may have and go straight for the mussels.


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With their uninhibited willingness to quickly gobble down flies, the brilliant coloration and tell tale cutthroat marks adorning the underside of its jaws; the cutthroat trout has to be one of the most sought after of all the trout species by fly anglers. The most famous of the cutthroat trout species is the Westslope cutthroat trout which was discovered in 1805 by Lewis and Clark below the great falls of the Missouri River. A few years after the original discovery the word had spread throughout the fly fishing community about these fish's spirited fighting ability and distinct coloration and the interest from fisherman never faded.
One of the main color characteristics that people associate the cutthroat with is two yellow/orange or red lines in the skin folds of the underside of the lower jaw. |
With their uninhibited willingness to quickly gobble down flies, the brilliant coloration and tell tale cutthroat marks adorning the underside of its jaws; the cutthroat trout has to be one of the most sought after of all the trout species by fly anglers. The most famous of the cutthroat trout species is the Westslope cutthroat trout which was discovered in 1805 by Lewis and Clark below the great falls of the Missouri River. A few years after the original discovery the word had spread throughout the fly fishing community about these fish's spirited fighting ability and distinct coloration and the interest from fisherman never faded.
About the Cutthroat
Cutthroat can be characterized as having short, conical heads with a somewhat pointed to rounded snout and a rather large mouth holding sharp developed teeth on both the upper and lower jaws. One of the main color characteristics that people associate the cutthroat with is two yellow/orange or red lines in the skin folds of the underside of the lower jaw. As far as the body color for these inland cutthroats is concerned it really depends on when and where you catch these fish. The colors can range from olive, yellow, to silver with orange to red highlights depending on what time of year it is. The coastal form of the cutthroat is colored differently than its inland brethren, with dark to olive-green coloration with numerous black spots.
Cutthroat, like all the other trout species, are an indicator species meaning they normally can only live in the cleanest, coldest waters. These rivers or lakes can be made up of fresh, brackish or salt water but some of the highest densities of inland species of these fish can be found in North America slightly west of the Rocky Mountains.
With the adult anadromous forms of cutthroat the return migration to their freshwater spawning streams begins in late autumn and early winter, but the actually spawning for both types of fish takes place somewhere between February and May. Spawning cutthroat will run high up their native rivers finding a quiet, gravelly, slow tail out location to begin the courtship. Once the males and females have paired the female prepares the redd, and deposits anywhere from 1,100-1,700 eggs. After the eggs have been fertilized, the tiny fry will develop and eventually hatch 6-7 weeks later. Water temperatures for this spawning process are generally around the 10 degree Celsius mark. The fry will usually emerge in July, which coincides with the peak productivity of the watershed they live in, giving them the best chance to feed and grow quickly. The average length of this species ranges from 12 to 15 inches and is generally one-half to 2 pounds. *** maturity occurs at an age between 2 and 4 years then the process repeats itself.
Fishing Techniques
The cutthroat trout has become regarded as one of the best species to target with a fly rod. Nestled away in astonishing landscapes and in some of the most spectacular rivers in North America the cutthroat trout entices all who love to fly fish. The real beauty of cutthroat trout is that they are a working-man's fish. Whereas browns and brookies tend to disappear in blue bird days with high sun, cutthroat trout are never too weary to come out and play. Additionally, if you are fishing for cutthroats, than there is no real need for those extra-early sunrise starts, which is very convenient for those of you working full time jobs. Nymph flies are a good place for anglers to start if they are fishing a new body of water, or fishing ahead of oncoming storms.
Cutthroat for some reason or other seem to key in on these subsurface presentations and strike nymphs greedily with the coming of inclement weather. Most often cutthroat are fished for and caught with dry fly patterns emulating stoneflies, caddis flies and mayflies. Anglers should look for typical holding cover as cutthroats are ambush predators. Good cover for cutthroat comes in the form of as eddies, log jams, large boulders, transitions between fast and slow water and oxygenated riffles. Over the last few years in heavily fished areas such as the Yellowstone National Park, cutthroat on certain days appear to become quite finicky, and tough to fool. Whether it is the years of catch & release, or just general malaise the large cutthroat will turn up their noses to everything you have to offer. Fortunately for anglers just as quick as this stubborn behavior had set in, it will depart and these large cutthroats for no reason will be attacking flies faster then you can cast them in the water.
Fly Selection
Cutthroat trout will eat just about anything that fly anglers can cast out onto a river quietly, making the group of effective flies used for these fish very large. Cutthroat like other trout will regularly consume a wide variety of aquatic insects and invertebrates but dabble a little with any unfortunate land insects like ants, beetles, and hoppers that happen to get blown into the water. With such a widely-ranging diet, choosing the right fly to use might seem tough but it really is not. Cutthroat trout are not discriminating feeders, if you do not match the hatch exactly, fish will still take interest in your flies. A good idea is to carry a few of the well known general imitator type patterns for nymphs and dries such as the hares ears and princes, to Hendrickson's and March Browns. Attractor patterns can also do a good job in catching fish especially when there is no sign of a hatch going to bloom that night. Streamer patterns have also shown good results with cutthroats. Generally these fish fall victim to wooly buggers, and leech type patterns tied with breathable materials. Basically, when fly fishing for cutthroat you want to try and match the hatch as best you can, but more importantly just get a fly out in front of these fish's faces and see if you are generating any interest.
Good Selection. A good idea is to carry a few of the well-known general imitator type patterns for nymphs and dries such as the hares ears. |
In you are going to be fishing cutthroats in the early spring, the resident aquatic insects will just start to get going at this time making using nymphs a very effective technique. Stoneflies and Caddis's are usually some of the best early action for fly anglers with Mayflies heating up soon after. A good point to remember is that with the spring months come dark colored insects so any nymph pattern using dark browns or black should suffice. As summer heats up the insects will become lighter in color and smaller in size so tailoring your efforts towards these types of bugs should produce handily. Your best bet when this heat of summer comes into play is to use dry fly patterns as much as you can. Cutthroats have a weakness for dead-drifted flies, and it can make for a great day out on the water watching fish rise readily after you're dry. Fishing dries can be especially effective if you are going to be fishing a pastureland stream. Winds over these grassy areas will frequently blow terrestrial insects into the water where the opportunistic cutthroat will rarely pass up an easy meal.
Later during the year cutthroats will continue to feed on nymph and dries if the weather is good but turn to feeding on small resident minnow imitations and egg patterns. Once the larger fish reach the redds and the spawn begins, eggs flies drifted slowly downstream in bright oranges and pinks will catch some good fish. At this time of the year most cutthroat steams will be closed so make sure that you check the local regulations to see where and how you are able to fish.
Weather & Sunlight
As was alluded to earlier, sunlight really does not play a very important role in the feeding habits of cutthroat trout. Whether it be sunny or overcast cutthroat will do as they always do, look to feed aggressively. Heavy rain can slow things up a bit by muddying the waters and making it hard to come into contact with fish. As the level of the river water rises due to rain the clarity will decreases making any fishing efforts unfruitful. Wait a few days until the level of the water starts to recede and the fish will once again become active. If you do decide to fish while the water is still a bit discolored, the key to consistently catching fish is to fish the water slow. Fish in these types of conditions will have a hard time seeing and accurately striking flies so giving them as much of a chance to get a good, long look at what you are offering works only to your advantage.
Cutthroat streams are visited by thousands of fly anglers from far and wide every year for a few main reasons; these fish are extravagantly colored, tough fighters and greedily feed on flies presented by even novice anglers. Getting the chance to fish these great trout does not have to be a trip of a lifetime, they are wide-spread through North America and one needs to just take the time a see what the rivers around them have to offer. Get out and let a few dries ride the water, it won't take long to get a few cutthroat interested in what you have to offer.
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Birth Name: Ryker Baloun
Birth Date: October 8, 2005 (18 years old)
Birth Date: October 8, 2005 (18 years old)
Nickname: Unknown
Birthplace: USA
Birthplace: USA
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Pack This: Canned Craft Beer
Not That: Bottled Craft Beer
It used be that blissed out festival hippies only had one option for quality beer — the reliable bottle. The thing about beer bottles is that they suck for camping. They're bulky and heavy and have a tendency to be shattered into oh so many unfriendly shards. Cans be smashed down, fit better in coolers, and fit way better into beer coozies.
For a more in depth rundown on the can versus bottle debate, check out our friends over at Liquor Snob who have the classy bottle and the trusty can battle it out for superiority (spoiler alert: cans win). They forgot one key point. You can't key a bottle and shotgun a beer man-style.


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Part of a very occasional series in which we improve odd descriptions of vanity press books advertised in The New York Times Book Review by adding the phrase "Wacky high jinks ensue" at the end of them. Why? Because we just want to help. Ad copy verbatim (except for the high jinks) from the April 29 issue. Odd punctuation should be considered (sic):
Plateau Ivory 10,000: Down With Destiny: Wishwanath, the Monarch of Plateau Ivory was driven to the edge by believing his father who had died of an accident was alive and following him to almost everywhere! Wacky high jinks ensue.Daaaad! Stop it!
Underneath a Flight Attendant talks about [name redacted]'s adventures, during her long flying career as a cabin crew and her musings as a spiritual guru and peace seeker. It's a biography that will blow your mind! Wacky high jinks ensue.Really, miss, all I asked for was a blanket.
"Now I Can Call Myself A Biker": This is the story of David's exploits and adventures to gain experience he so desperately wanted. It has shown that even at his age, in his mid-life crisis, it wasn't too late to learn how to ride a motorcycle. Wacky high jinks ensue.Oh, big deal. I just bought a seven-speed bicycle. Now, that takes guts.


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Page Not Found!
Sorry! We could not find the specific page you requested. Pastor's Den/March Month 2011 Please check the address and try again. If you still cannot find the page you are looking for, try going to the top level "www.bethlehemofbonham.net" and clicking on a link from the menu. |


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Overview of the Common UNIX Printing System
- Overview of the Common UNIX Printing System
- The User's Perspective
- The Developer's Perspective
- Resources
Overview of the Common UNIX Printing System
Printing within UNIX has historically been done using one of two software packages: the Berkeley Line Printer Daemon (LPD) and the AT&T Line Printer (LP) system. These print spoolers were designed in the 1970's only for printing text to line printers. Over the years vendors have tried adding support for other types of printers with mixed success, and replacements like LPRng are little more than a new coat of paint over an aging text spooling interface.
The Common UNIX Printing System (CUPS) is a completely new printing environment which is designed to handle printing of modern document files to today's printers while supporting existing printing commands and applications transparently.
On the outside, CUPS is built upon the Internet Printing Protocol (IPP) which provides advanced network/Internet printing capabilities. On the inside, CUPS provides an extensible system interface that allows new printers, document formats, and interfaces to be supported quickly, easily, and transparently.
The Administrator's Perspective
Printing used to be the bane of every system administrator. With CUPS, the administrator immediately has command-line, WWW, and GUI interfaces to the print server and printer drivers. Point your Web browser at the print server's administration page and you'll have the pleasure of configuring printers using simple wizards, which guide you through every step of the process. If you prefer to do things from the command line, CUPS provides two simple tools for adding and configuring your printers.
Imagine setting up a laser printer queue with a simple command such as
lpadmin -p DeptLaserJet -v ipp://22.214.171.124/ipp -E -m laserjet.ppd
This simple command configures a networked HP LaserJet printer called "DeptLaserJet." The printer is on the network and using IPP ("-v ipp://184.108.40.206/ipp"), is enabled by default ("-E"), and uses the sample HP LaserJet printer driver supplied with CUPS ("-m laserjet.ppd").
If you have multiple "client" computers that will be printing through your print server, CUPS even makes that job easy; just add the broadcast address for your network to the CUPS configuration file, for example:
BrowseAddress 126.96.36.199
Once configured, all clients on the same subnet will automatically see the printers you have defined on the server. You don't have to configure every client, or use unreliable schemes to pull or push configuration files on the client machines, you just let CUPS do the work for you!
If you have a complex network with multiple subnets, you just need to set up a few clients to relay the printer information from the server to the local subnets. The clients (or other servers) merely act as repeaters for the main server, again limiting the amount of work you have to do.
This automatic printer sharing offers another additional feature not found in any other printing solution: implicit classes. Implicit classes are groups of identical network printers that each client creates automatically. When a user prints to an implicit class, the client chooses the next available server in the list and forwards the job for printing. If a server is overloaded or goes down, the client will use another server automatically. Thus, implicit classes provide automatic load balancing and fail-safe operation, and you don't have to do a thing!