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A method of isolating a monoclonal antibody capable of inhibiting any one of IL-3, GM-CSF and IL-5 binding to the common receptor βc, or a monoclonal antibody capable of inhibiting the cytokines binding to a receptor analogous to βc. The method includes the steps of immunizing an animal with a cytokine receptor or portion of a cytokine containing the critical binding site which portion includes the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof. Antibodies producing cells from the animal are then isolated and fused with a myeloma cell line and then screened for a cell line that produces an antibody of the desired type. A monoclonal antibody, or fragments thereof capable of inhibiting the binding of the cytokines IL-3, GM-CSF and IL-5 to the βc receptor, and a hybridoma cell line producing the antibody are also claimed.
1. A method of inhibiting IL-5, IL-3 or GM-CSF mediated proliferation of a chronic myelomonocytic cell by contacting said cell with a monoclonal antibody BION-1 or a fragment thereof capable of inhibiting binding of cytokines IL-3, GM-CSF or IL-5 to the common receptor βc, wherein the monoclonal antibody or fragment thereof binds to both the B′-C′ loop and the F′-G′ of domain 4 of the βc subunit, wherein said contacting results in inhibition of IL-5, IL-3 or GM-CSF mediated proliferation of said cell.
2. A method of inhibiting IL-5, IL-3 or GM-CSF mediated eosinophil activation, eosinophil production or eosinophil survival, by contacting an eosinophil with a monoclonal antibody BION-1 or a fragment thereof capable of inhibiting the binding of cytokines IL-3, GM-CSF or IL-5 to the common receptor βc, wherein the monoclonal antibody or fragment thereof binds to both the B′-C′ loop and the F′-G′ of domain 4 of the βc subunit, wherein said contacting results in inhibition of IL-5, IL-3 or GM-CSF mediated activation, production or survival of said eosinophil.
This application is a divisional of U.S. application Ser. No. 09/762,963 filed Feb. 14, 2001, now U.S. Pat. No. 6,720,155 which is a 35 U.S.C � 317 of a PCT/AU99/00659, which claims priority from Australia PP5251, filed Aug. 13, 1998. The entire disclosure and contents of the above patents and applications are hereby incoporated by reference.
This invention relates to a method of isolating monoclonal antibody inhibitors and reagents derived therefrom and other inhibitors of cytokine binding including monoclonal antibodies and reagents derived therefrom and small molecules capable of inhibiting binding of GM-CSF, IL-3 and IL-5 to the common beta receptor subunit.
Human interleukin (IL)-5, IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are cytokines involved in hemopoiesis and inflammation (Metcalf; 1986). All three cytokines stimulate eosinophil production, function and survival (Metcalf; 1986) and therefore have the ability to influence inflammatory diseases such as asthma, atopic dermatitis and allergic rhinitis where the eosinophil plays a major effector role. IL-5, being the eosinophil specific cytokine, has received most of the initial attention with IL-5 mRNA and protein levels noted to be elevated in lung tissue and bronchoalveolar lavage (BAL) fluid from symptomatic asthma patients (Fukuda et al 1994). Correlation between IL-5 levels and allergen challenge and disease activity have also been seen (Sur et al, 1996). It is becoming apparent, however, that not only IL-5 but also GM-CSF and IL-3 play a role in eosinophil production and activation in asthma as there is evidence of both GM-CSF and IL-3 being synthesized at sites of allergic inflammation (Bagley et al, 1997b; Allen et al 1997). It is possible that expression of these cytokines contributes to the total number of infiltrating eosinophils and the degree of eosinophil activation. Alternatively, they may be responsible for different phases of eosinophil infiltration. Recent kinetic data from patients undergoing antigen challenge showed that IL-5 levels increased between days 2-7 post challenge, whilst GM-CSF peaked at day 2, and remained elevated throughout day 16. Furthermore, GM-CSF detection extended beyond the site of allergen challenge.
IL-S, GM-CSF and IL-3 stimulate eosinophils and other normal and cancer cells by binding to cell surface receptors that comprise a ligand-specific α chain and a β chain which is shared by the three receptors (βc) (Bagley et al 1997a). Binding to each receptor α chain is the initial step in receptor activation, however, engagement of either α chain alone is not sufficient for activation to occur. Recruitment of βc by each ligand: α chain complex follows, a step that has two major functional consequences: firstly, it allows the binding of IL-5, GM-CSF and IL-3 to become essentially irreversible; and secondly, it leads to full receptor activation (Bagley et al 1997a). Since βc is the major signalling component of these receptors its engagement leads to the activation of JAK-2, STAT-5 and other signalling molecules culminating in the full plethora of cellular activities commonly associated with either IL-5, GM-CSF and IL-3 stimulation such as eosinophil adherence, priming for degranulation and cytotoxicity, and prolongation of viability (Bates et al, 1996).
In order to block or antagonize the activity of eosinophil-activating cytokines in vivo three major approaches are being tried. One of them utilizes antibodies to the implicated cytokines. For example, antibodies to IL-5 are being used in an animal model of allergen-induced asthma and have shown to have a relatively long lasting effect in preventing eosinophil influx into the airways and bronchial hyperresponsiveness (Mauser et al, 1995). A second approach relies on IL-5 or GM-CSF mutants which can bind to the respective α chains with wild type affinity but which have lost or shown reduced ability to interact with human βc. IL-5 mutants such as E13Q, E13K and E13R, and the human GM-CSF mutant E21R directly antagonize the functional activation of eosinophils by IL-5 or GM-CSF respectively (Tavernier et al 1995; McKinnon et al 1997; Hercus et al 1994b). However, at least in the case of E13K, eosinophil survival is not antagonized and in fact this mutant is able to support eosinophil survival (McKinnon et al 1997). A third approach involves the use of soluble receptor α chains which can sequester circulating cytokines. However, this carries the risk of a cytokine:receptor α chain complex potentially interacting with surface-expressed βc and triggering receptor activation. The common theme amongst these approaches is that they tackle a single receptor system involving either IL-5, GM-CSF or IL-3 leaving the other two eosinophil-acting cytokines unaffected. Although the concomitant administration of IL-5 and GM-CSF antagonists may be considered, this may be clinically impracticable.
An alternative approach to blocking eosinophil-activating cytokines involves targeting the common β chain of their receptors. Although βc does not directly bind IL-5, GM-CSF or IL-3 alone, it does bind to these cytokines complexed to the appropriate receptor α chain. Lopez et al in WO 97/28190, which is incorporated herein by reference in its entirety, have identified the major binding sites of, βc for the IL-5:IL-5Rα, GM-CSF:GM-CSFRα and IL-3:IL-3Rα complexes. Significantly, these sites are utilized by all three complexes and comprise the predicted B′-C′ loop and F′-G′ loop in βc. Thus targeting βc is not only desirable but also feasible, with the added potential to allow the simultaneous inhibition of IL-5, GM-CSF and IL-3 action by a single agent. These workers have shown that certain mutants in the B′-C′ and the F′-G′ loop fail to bind IL-5, GM-CSF and IL-3.
The present invention results from the isolation of a monoclonal antibody BION-1) raised against the membrane proximal domain (domain 4) of βc which is able to block the production and activation of human eosinophils stimulated by IL-5, GM-CSF or IL-3 and blocks the growth of leukaemic cell lines. This MoAb was able to block the high affinity binding of all three cytokines to eosinophils by binding to residues in the predicted B′-C′ and F′-G′ loops of βc, and prevented receptor dimerization and βc phosphorylation. It was found that raising an antibody capable of blocking the binding of all three cytokines was possible by screening monoclonal antibody-expressing hybridoma cell lines arising from immunising mice with cells expressing only domain 4 of βc and lacking domains 1 to 3 and expressing domain 4 and the transmembrane and cytoplasmic regions.
Additionally this finding is likely to have implications for other members of the cytokine receptor superfamily some of which are shared subunits in a given subfamily (that is they bind several cytokines), and some which are ligand specific and bind to only one cytokine. The receptor α-chains for GM-CSF, IL-3 and IL-5 and βc belong to the rapidly expanding cytokine receptor superfamily. Within this superfamily several sub-families are now emerging that are characterized by the sharing of a communal receptor subunit by multiple ligands: gp 130 acts as an affinity converter and signal transducer for IL-6 (Hibi et al., 1990; Taga et al., 1992), IL-11 (Hilton et al., 1994), oncostatin M (Liu et al., 1992), ciliary neurotrophic factor, leukaemia inhibitory factor (LIF) (Ip et al., 1992) and cardiotrophin-1(Pennica et al., 1995); the LIF receptor (LIFR) also binds ciliary neurotrophic factor (Davis et al., 1993), cardiotrophin-1 (Pennica et al., 1995) and oncostatin M in addition to LIF (Gearing et al., 1994); IL-2R β supports affinity conversion and signalling of IL-2 and IL-15 (Giri et al., 1994); IL-2R γ chain affinity converts IL-2 (Takeshita et al., 1992), IL-4 (Russell et al., 1993), IL-7 (Noguchi et al., 1993), IL-9 (Kimura et al., 1995) and IL-15 (Giri et al., 1994); evidence also suggests that IL-4 and IL-13 share a receptor component (Zurawski et al., 1993) and this subunit has recently been cloned (Hilton et al., 1996). It is not known which residues in gp 130, LIFR and IL-2R β and γ chains are important for ligand binding or indeed whether different ligands share or have unique sets of binding determinants on these communal receptor subunits. Because these common subunits are vital for transducing signals by several ligands, the possibility arises that interfering with the ability of these common subunits to bind ligand or to form homodimers may affect the action of more than one ligand.
Clear similarities in structure between βc and other cytokine receptors have been recognised and similarities in at least part of the binding site, the F′-G′ loop, have been identified in Lopez et al in WO 97/28190. Accordingly it is an expectation that the means employed by the inventors to obtain a monoclonal antibody that inhibits binding of IL-3, GM-CSF and IL-5 will also lead to the isolation of monoclonal antibodies that inhibit binding of other cytokines to their respective receptors.
In a broad form of a first aspect the invention could be said to reside in a method of isolating a monoclonal antibody capable of inhibiting any one of IL-3, GM-CSF and IL-5 binding to the common receptor βc, or a monoclonal antibody capable of inhibiting a cytokines binding to a receptor analogous to βc, said method comprising the step of immunising an animal with a cytokine receptor or portion of a cytokine receptor containing the critical binding site which portion might include the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof, isolating antibody producing cells from said animal and fusing antibody producing cells with a myeloma cell line, screening for a cell line that produces an antibody of the desired type.
The immunisation may involve introducing a cDNA clone of a portion of or all of the common receptor including the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof, into a cell and proliferating said cells to form a recombinant cell line, inoculating an animal with said recombinant cell line, isolating antibody producing cells from said animal and fusing the antibody producing cell line with a myeloma cell line to form a hybridoma cell line, screening for a hybridoma cell line that produces an antibody that binds to the recombinant cell line but not to the parent, and then testing for inhibition against all three cytokines. In one form the cell into which the cDNA clone is introduced is mammalian and one commonly used mammalian cell line is a COS cell.
The cDNA may encode a full or partial portion of domain 4 when it is in a configuration where the F′-G′ loop and/or the B′-C′ loop is in its native shape. The data below show that cDNA encoding substantially only domain 4 of the extracellular portion of βc as well as the transmembrane and the intracellular portions maintains these sites in a sufficiently integral conformation so that an antibody raised thereagainst will give the inhibition sought. It is postulated that the same will be the case for analogous receptors for the cytokine superfamily. This method should be distinguished from immunising with the whole receptor since the extracellular domain 4 is likely to be covered or masked by other domains in the whole receptor.
βc has two repeats of the cytokine receptor module (CRM), each of which has two discrete folding domains (CRDs), so that in total βc has 4 domains hence named domains 1 to 4 (β1 to 4). It is postulated that domain 2 of any CRM may be an equivalent of domain 4 and therefore domain 2 may be used in the immunisation.
In addition the domain 4 of βc or equivalent domain in other cytokine receptors may be expressed in isolation in a microbial host such as Escherichia coli and used to immunise animals for developing monoclonal antibodies.
The analogous receptor may be any one of the cytokine superfamily receptors but not limited to the group comprising βc, LIFR, gp130, IL-2Rβ, IL-4R/IL-13R, IL-2Rγ, IL-3Rα, EPOR, TPOR and OBR.
It will be understood that in one specific form of this aspect of the invention the method is used to isolate a monoclonal antibody that inhibits cytokine binding to a common receptor subunit. The common receptor is envisaged to be selected from the group of receptors acting for more than one cytokine including but not limited to gp130, LIFR, IL2Rβ/IL2Rα, and EL-4R/IL-13R in addition to βc.
It will also be understood that the invention encompasses monoclonal antibodies or fragments thereof produced as a result of this first form of the invention.
In a broad form of a second aspect the invention could be said to reside in a monoclonal antibody, or fragments thereof capable of inhibiting the binding of the three cytokines IL-3, GM-CSF and IL-5 to the βc receptor.
The degree of inhibition may range from complete inhibition to moderate inhibition, which inhibition will of course be dependent on the amount of monoclonal antibody or fragments thereof added to inhibit and the relative affinity of the antibody or fragment thereof to the βc.
The extent of inhibition of respective ones of the three cytokines is not necessarily identical and may vary, so the different cytokines may be inhibited from binding to different degrees.
The antibody fragments may be larger portions such as Fab fragments or much smaller fragments of the variable region. These fragments may be used as separate molecules or alternatively may form part of a recombinant molecule which is then used for therapeutic purposes. Thus for example the monoclonal antibody may be �humanised� by recombining nucleic acid encoding the variable region of the monoclonal antibody with nucleic acid encoding non-variable regions of human origin in an appropriate expression vector.
The inhibition preferably leads to blocking of at least one function of all three cytokines. One of the benefits that is proposed to be derived from these antibodies or antibody fragments is their use in modifying cells stimulated by one of the three cytokines, and more in one specific form modifying the activity of the three cytokines is proposed to impact greatly on eosinophil function. Therefore preferably the activity leads to inhibition of stimulation of effector cell activation and where the antibody or fragment thereof is to be used for treatment of asthma leads most preferably to inhibition of IL-5, IL-3 & GM-CSF mediated eosinophil activation. It will be understood however that cells other than eosinophils are also the effectors of adverse conditions in humans and animals as a result of stimulation by these cytokines and inhibition of such stimulation is also contemplated by this invention. These include cells that express either one or all of GM-CSF, IL-3 and IL-5 receptors, the stimulation of which leads to pathology. Examples of these are leukaemic cells, endothelial cells, breast cancer cells, prostate cancer cells, small cell lung carcinoma cells, colon cancer cells, macrophages in chronic inflammation such as rheumatoid arthritis, dendritic cells for immunosuppression and neutrophils in inflammation.
Thus in one form the invention may be said to reside in an inhibitor of leukaemic cell growth wherein the inhibitor is capable of inhibiting the binding of one or all of IL-3, GM-CSF and IL-5 to the βc receptor. The inhibitor may be BION-1 or an agent capable of inhibiting BION-1 binding with βc.
A number of different facets of eosinophil function might be modified so that in one form IL-5, IL-3 & GM-CSF mediated eosinophil survival is inhibited or blocked. In a second form IL-5, IL-3 and GM-CSF mediated eosinophil activation is inhibited or blocked.
In one form of this second aspect of the invention the monoclonal antibody or fragment thereof binds to at least the F′-G′ loop of domain 4 of the βc subunit.
In an alternative form the monoclonal antibody or fragment thereof binds to at least the B′-C′ loop of domain 4 of the βc subunit but this alternative form is not limited to monoclonal antibodies or fragments thereof that only bind to the F′-G′ loop but includes monoclonal antibodies or fragments thereof that perhaps binds to both the F′-G′ as well as the B′-C′ loop of domain 4 of the βc.
It is thought that the monoclonal antibody isolated by the inventors inhibits dimerisation of the common receptor units and thus the invention might encompass an antibody or fragments thereof of the second aspect of the invention that inhibit βc receptor dimerisation.
In one very specific form the monoclonal antibody is the antibody produced by the hybridoma cell line BION-1 (ATCC HB-12525).
In a broad form of a third aspect the invention could be said to reside in a hybridoma cell line capable of producing a monoclonal antibody of any form of the first or second aspect of the invention.
In one specific form of the third aspect of the invention the hybridoma cell line is BION-1 (ATCC HB-12525).
Since GM-CSF, IL-3 and IL-5 need to bind their respective a chains before being able to interact with βc, at present most screening for new inhibitors utilise cell-based assays where both, α and βc receptor units are co-expressed. Solid phase assays rely on inhibition of GM-CSF, IL-3 or IL-5 to their respective a chain only since these cytokines cannot bind to βc alone. Since BION-1, unlike these three cytokines, can directly bind to βc we propose that it can be used as a novel solid phase screening assay. Any compound that binds the appropriate site which is likely to inhibit all three cytokines will also inhibit the binding of BION-1. Additionally once further inhibitory compounds are uncovered these could be used in the place of BION-1 in that screening process. This therefore facilitates the screening of larger number of candidate inhibitor compounds.
In a broad form of a fourth aspect therefore the invention could be said to reside in a method of screening peptides, oligonucleotides and other small molecules for their capacity to competitively inhibit the binding of BION-1 or the binding of an agent capable of inhibiting BION-1 binding, to the βc subunit.
Generally the screening assay involves contacting BION-1 or fragment thereof with the βc subunit or fragment thereof as well as a candidate inhibitory compound, and measuring the degree of binding.
A reporting means is preferably provided to facilitate the detection of binding of BION1 or fragment thereof with βc subunit or fragment thereof. Thus, for example, a competitive binding assay using labelled BION-1 could be used for this purpose. βc or domain 4 of βc is immobilized on a plate or tube and several compounds added, followed by labelled or tagged BION-1 or fragments thereof. Since BION-1 binds the region of βc involved in binding all three cytokines, any compounds that block or reduce the binding of BION-1 or fragments thereof to βc or domain 4 will be considered candidate inhibitory compounds. Thus, the availability of BION-1 as an agent that for the first time allows the direct binding to the cytokine binding region of βc affords a novel test for the identification of simultaneous inhibitors of GM-CSF, IL-3 and IL-5. It will be understood that the same will apply for other cytokines and their respective receptors.
It will be understood that not the entire βc subunit needs be used to screen candidate compounds, and certainly the present data indicates that a fragment of the βc subunit encompassing domain 4 has sufficient structure in common with the native βc subunit to reflect the configuration of the cellular target for an inhibitor useful for an in vivo effect.
In a broad form of a fifth aspect, the invention could be said to reside in a cytokine inhibitor capable of simultaneously blocking the binding of βc by IL-3, GM-CSF, and IL-5 made according to the fourth aspect of the invention.
It is thought that compounds that inhibit binding of the IL-3, IL-5 and GM-CSF to the βc will be therapeutically useful for intervention in conditions where IL-3, GM-CSF and IL-5 play a pathogenic role, mainly allergy, asthma, acute and chronic myeloid leukaemias, lymphoma and inflammation including rheumatoid arthritis, breast cancer and prostate cancer.
Similarly for other common cytokine receptors it is thought that antagonists or agonists will be therapeutically useful. gp130 is functionally analogous to βc in that it is a common binding sub-unit and signal transducer for the IL-6, oncostatin M (OSM), ciliary neutrotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and IL-11. It is suggested that raising an antibody against a domain analogous to domain 4 of βc will also lead to blocking of two or more of these cytokines. Antagonism of this receptor system will be useful in inflammation, leukaemia and lymphoma. Antagonists to IL2Rβ/IL2Rα may be useful as immunosuppresants. Antagonists of LIFR may be useful for the prevention of implantation of embryos in uteri. Antagonists of IL4/L-13 will inhibit IgE production and may be useful in treating asthma and allergies.
Materials and Methods
ΔQP cDNA: To express domain 4 of βc on the cell surface we cloned the activated βc mutant, hβcΔQP, with an extracellular deletion removing domains 1 to 3 (D'Andrea et al 1996), into the eukaryotic expression vector pcDNA3 (Invitrogen).
Cytokines and cell lines: Recombinant human IL-3 and GM-CSF were produced in E. coli as described (Barry et al 1994, Hercus et al 1994b). Recombinant human IL-5 was purified from E. coli by Bresatec (Adelaide, South Australia). Recombinant EPO was purchased from Johnson & Johnson (New Jersey). TNFα was a gift from Dr. J. Gamble in the Hanson Centre for Cancer Research. COS cells were transfected with receptor cDNA as described previously (Woodcock et al 1994). CHOβc and CHOΔQP cells stably expressing either full length βc or domain 4 respectively were generated by electroporation (Hercus et al, 1994a). TF1.8 cells were a gift from Dr J. Tavernier from University of Gent, Belgium. MO7e cells, a human megakaryoblastic cell line, were from Dr P Crozier, Aukland, New Zealand. Human eosinophils were purified from the peripheral blood of slightly eosinophilic volunteers via sedimentation through dextran and centrifugation through a discontinuous density gradient of hypertonic Metrizamide, as previously described (Vadas et al 1979). Eosinophils were more than 92% pure. Human neutrophils and monocytes were purified from peripheral blood as described previously (Lopezet al, 1990) with more than 95% purity.
Generation of anti-βc MoAbs: BALB/c mice were immunized intraperitonally with 1�107 COS cells transfected with βc or ΔQP expression constructs. ΔQP constructs express substantially only domain 4 of the extracellular domains of βc. The immunizations were repeated 4 times at two-weekly intervals. Four weeks after the final immunization, a mouse was boosted with 2�106 COS transfectants intravenously. Three days later, splenocytes were harvested and fused with NS-1 myeloma cells as previously described (Sun et al 1996). Hybridoma supernatants were screened on CHO βc or CHO ΔQP cells by flow cytometry, with untransfected CHO cells as a control. All antibodies were from single hybridoma clones as selected by limiting dilution method. MoAbs were purified from ascites fluid or hybridoma supernatant by a protein A sepharose column. The isotypes of MoAbs were tested with a Mouse MoAb Isotyping Kit (Boehringer Mannheim, Germany). Fab fragments were generated using a Fab Preparation kit (Pierce, Rockford, Ill.) following the supplied protocol.
Immunofluorescence: Freshly purified neutrophils, eosinophils, monocytes, or CHO and COS cell transfectants (5�105) were incubated with 50 μl of hybridoma supernatant or 0.25 mg of purified MoAb for 45-60 min at 4� C. Cells were washed twice and then incubated with FITC-conjugated rabbit anti-mouse Ig (Silenus, Hawthorn, Victoria, Australia) for another 30-45 min. Cells were then washed and fixed before analysing their fluorescence intensity on an EPICS-Profile II Flow Cytometer (Courter Electronics). Two colour staining was carried out by additional incubation with another MoAb directly coupled to PK.
Ligand Binding Assay: IL-3 and GM-CSF were radio-iodinated by the iodine monochloride method (Contreras 1983). 125I-IL-5 was purchased from Dupont NEN (North Sydney, NSW, Australia). Binding assays were performed as previously described (Lopez et al 1989). Briefly, 1-2�106 TF-1.8 cells were preincubated with BION-1 Fab fragments, anti-βc or control MoAbs over a concentration range of 0.06 to 4200 nM for 1 hour. Radio-labelled ligand was then added and incubated for a further two hours before the cells were separated from free label by spinning through FCS. Counts associated with the resulting cell pellets were determined by counting on a γ counter (Cobra Auto Gamma; Packard Instruments Co, Meridien, Conn.). Non-specific binding was determined for each ligand by binding in the presence of a 200 fold excess of unlabelled cytokine.
MoAb binding assay: MoAbs were radio-iodinated by the chloramine-T method (McConahey 1980). Saturation binding studies were performed by incubating 2�106 TF1 cells in a range of concentrations of radio-labelled antibodies in the presence or absence of excess unlabelled antibodies. The binding affinity of each anti-βc MoAb to its antigen was determined by Scatchard transformation (Scatchard 1949) and analysed with the ligand program (Munson and Rodbard, 1980). Competition binding experiments were set up by preincubating the TF1.8 cells with a range concentration of IL-3, or GM-CSF, or IL-5 prior to adding radio-labelled MoAb QP1 for two hours as per ligand binding assay. Epitope analysis was determined by testing the capacity of each unlabelled MoAb to compete for the binding of each radio-labelled MoAb to the βc on COS cell transfectant.
Co-immunoprecipitation of α and β chains and the βc phosphorylation assays: M07e cells were surface labelled with 125I by the lactoperoxidase method as described previously (Walsh and Crumpton, 1977). The labelled cells incubated in either medium containing IL-3 (100 ng/ml) alone or IL-3 together with the MoAb QP1, 1C1 (0.5 mg/ml) or 7G3 (30 mg/ml) for 5 min. Cells were lysed in lysis buffer consisting of 137 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10% Glycerol, 1% Nonindet P40 with protease and phosphatase inhibitors (10 mg/ml leupeptin, 2 mM phenylmethlysulphonyl fluoride, 10 mg/ml aprotonin and 2 mM sodium vanadate) for 30 min at 4� C. followed by centrifugation of the lysate at 10,000�g for 15 min to remove cellular debris. The lysate was precleared with mouse-Ig-coupled Sepharose beads for 18 h at 4� C. and incubated with anti-IL-3Ra, anti-βc MoAb beads for 2 hr at 4� C. The beads were washed 6 times with lysis buffer and immunoprecipitated proteins were separated by SDS-PAGE under reducing condition. The immunoprecipitated proteins were detected by a Phosphorlmager (Molecular Dynamics, Sunnyvale, Calif.). The gels were then reprobed by Western blotting analysis with an anti-phosphotyrosine MoAb, 3-365-10 (Boehringer Mannheim, Frankfurt, Germany).
TF-1.8 cell proliferation assay: TF-1.8 cells were grown in the presence of 2 ng/ml of GM-CSF. The cells were starved for 24 hours before setting up proliferation assays as described previously (Sun et al., 1996). From dose-response curves the half-maximal proliferation dosage of IL-3 (0.3 ng/ml), GM-CSF (0.03 ng/ml), IL-5 (0.3 ng/ml) or EPO (5 ng/ml) was chosen to perform proliferation experiments in the presence of a range of concentrations of MoAbs. The 3H-Thymnidine incorporation of each sample was determined by liquid scintillation and expressed as disintegrations per minute (DPM).
Eosinophil survival assays: The maximal dose of IL-5 required to support eosinophil survival after 36 hours was determined. Eosinophils were then cultured with 1 nM of IL 5 plus anti-βc MoAbs for 36 hours. The viability of eosinophils was quantitated by propidium iodide staining and flow cytometry analysis as described (Nicoletti, 1991).
CD69 expression: CD69 expression on eosinophils was-measured by means of an antiCD69 monoclonal MoAb coupled to PE by flow cytometry.
βc mutants and MoAb Mapping: Single amino acid substitutions in the B′-C′ and F′-G′ loops of domain 4 of the βc have been described previously (Woodcock, et al., 1994; 1996). The cDNAs for wild type βc and each of the βc mutants in the B′-C′ and F′-G′ loops were introduced into COS cells by the electroporation (Hercus et al., 1994). Cell transfectants were analysed for surface expression with 48 hours after transfection. Mutants on the B′ and C′ β-strands such as L356N, W358N, 1374N and Y376N were expressed on FDCP1 cells from retroviral expression constructs (Jenkins et al., 1995). Epitope-mapping of anti-sc antibodies was analysed by Immunofluorescent study. The anti-βc MoAbs were tested for their abilities to recognise wild type βc and the βc mutants analysed by flow cytometer using standard immunofluorescence method. For each mutant, the experiment was repeated at least twice.
BION-1 binding inhibitory peptides: E. coli derived soluble βc domain 4 (sβc#4) was coupled to Maxisorp ELISA plates at 10 μg/1 ml in 0.1M carbonate buffer overnight and then blocked with 1% BSA. B45.pep (FHWWWQP-GGGCDYDDDK) was derived from four rounds of biopanning the Ph.D-7mer library with sβc#4 using an acid eluant. YB12.pep (FPFWYHAHSPWS-GGGCDYKDDDK) was derived from biopanning the Ph.D-12mer library with sβc#4 using an acid eluant. B45 was allowed the bond to sβc#4 at 0.0125 μM and YB12 was allowed to bind sβc#4 at 0.025 μM. The plate was washed in TBS+0.5% Tween. BION-1 was added to the plates at a starting concentration of 5 μg/ml and serial dilutions were used to titrate the BION-1 down to 0.004 μg/ml.
The plate was washed again and BION-1 binding to sβc#4 was detected with α-mouse conjugated to HRP, using a colour based reaction which was read on a plate counter by absorption.
BION-1 inhibition of chronic myelomonocytic cells: Peripheral blood from a patient with chronic myelomonocytic leukemia was centrifuged over Ficoll-Paque to separate the mononuclear cells. After washing and counting, the cells were plated on agar as a concentration of 105 per plate. After incubation in medium containing monoclonal antibodies BION-1 or 1C1, with or without IL-3, for 14 days at 37� C. the number of arising colonies were counted by mycroscopical examination. Each cell cluster containing more than 40 cells was counted as a colony.
Development of MoAb BION-1
Previous experiments have shown that the putative F′-G′ loop of βc contains a common binding site for IL-5, GM-CSF and IL-3 (Woodcock et al, 1996; WO 97/28190). We have now produced a blocking compound, represented by MoAb BION-1, by immunizing mice with COS cells transfected with a cDNA coding for domain 4 of βc. Screening of hybridoma supernatants was performed on a CHO cell line expressing domain 4 of βc. One hybridoma cell line was identified which produced a MoAb which specifically recognized this cell line and not a parental CHO cell line not expressing domain 4 of βc. This MoAb was termed BION-1 and was characterized in biochemical, binding and biological experiments.
BION-1 Recognizes Domain 4 as Well as Wild Type βc.
MoAb BION-1 was tested for reactivity against cell lines transfected with βc and against primary cells known to express IL-5, GM-CSF and IL-3 receptors. BION-1 recognized COS cells transiently transfected with βc CHO cells permanently transfected with βc, the erythroleukaemic TF-1 cell line, and purified peripheral blood human neutrophils, eosinophils and monocytes (
The antigen recognized by BION-1 was confirmed to be domain 4 of βc, by biochemical analysis of transfected cells.
BION-1 Inhibits the High Affinity Binding of IL-5. GM-CSF and IL-3 to TF-1 Cells and to Human Eosinophils
Given that domain 4 of βc is crucial for the high affinity binding of IL-5, GM-CSF and IL-3, we examined whether BION-1 was able to affect this binding. We found that BION-1 inhibited in a dose-dependent manner the binding of 125I-IL-5, 125I-GM-CSF and 125I-IL-3 to the human erythroleukaemic cell line TF-1. For each radioligand we used the smallest possible concentration to maximize the possibility of measuring high affinity. This can be more readily achieved with IL-3 and GM-CSF for which the difference between the low affinity component (provided by each α chain alone) and the high affinity component (provided by co-expressing βc with each a chain) is about 1,000 fold and 30 fold respectively. In the case of IL-5, the affinity conversion of βc is only in the 25 fold range, hence, high and low affinity binding cannot be clearly separated. This is likely to explain why BION-1 shows complete inhibition of 125I-GM-CSF and 125I-IL-3 binding (
The blocking effect of BION-1 was seen whether the MoAb was used as purified IgG or as Fab′ fragment.
Since one of the major clinical utilities of blocking IL-5, GM-CSF and IL-3 binding is likely to be in asthma, a disease in which eosinophils are believed to play a major role, it was important to test whether BION-1 could block the binding of IL-5, GM-CSF and IL-3 to these cells. As shown in
Epitope Mapping of BION-1
The fact that BION-1 inhibited the binding of 125I-IL-5, 125I-GM-CSF and 125I-IL-3 to TF-1 cells and eosinophils suggested that it might be binding to the critical region in βc to which these cytokines bind or at least in close proximity to it. To try to define the region/epitope in βc recognized by BION-1, we used several mutants of βc and examined whether substitutions of individual amino acids in the predicted B′-C′ loop or F′-G′ loop impaired BION-1 binding. Two sets of experiments were carried out. In the first instance we immunoprecipitated wild type βc from transfected COS cells with a MoAb anti-βc. The immunoprecipitates were then tested for reactivity with the control anti-βc MoAb 1C1, or BION-1. The results shows that βc mutants carrying the substitutions M363A/R364A, or E366A or R418A were not recognized by BION-1 (
BION-1 and IL-3 Reciprocally Inhibit Each Other's Binding
To confirm that the epitope recognized by BION-1 was the same or close to the binding site utilized by IL-5, GM-CSF and IL-3, we performed the reverse experiment, in which BION-1 was radio-labelled and increasing concentrations of IL-3 used to compete for 125I-BION-1 binding. The results showed (
BION-1 Specifically Inhibits the Function of IL-5. GM-CSF and DL-3 Including Their Stimulation of Eosinophil Production and Activation.
To ascertain whether the inhibition of IL-5, GM-CSF and IL-3 binding by BION-1 was translated into inhibition of IL-5, GM-CSF and IL-3 stimulation we used the factor dependent TF-1 cell line. This cell line proliferates in the presence of either IL-5, GM-CSF, IL-3 or erythropoietin (EPO) (
Titration experiments showed that BION-1 inhibited cytokine-mediated TF-1 cell proliferation in a dose-dependent manner with an ED50 of about 100-300 nM (
Since eosinophils are believed to be the major effector cells in asthma and they respond to IL-5, GM-CSF and IL-3, we examined BION-1 for its ability to block eosinophil production, eosinophil survival and eosinophil activation in response to these three cytokines. We found that BION-1 but not MoAb 8E4 inhibited the ability of IL-5, GM-CSF and IL-3 to stimulate the formation of eosinophil colonies from human bone marrow cells (Table II).
Importantly, BION-1 inhibited the pro survival activity of IL-5, IL-3 and GM-CSF on purified peripheral blood human eosinophils. Whilst these cytokines are essential for maintaining eosinophil viability (
BION-1 Specifically Inhibits IL-3 Receptor Dimerization and Activation
In order to define the mechanism of BION-1 antagonism we examined BION-1 for its ability to influence receptor dimerization and activation. We have previously shown that IL-3 or GM-CSF or IL-5 induce dimerization of the respective a chains with βc, a phenomenon that leads to receptor activation as measured by tyrosine phosphorylation of βc. This is confirmed here, with
BION-1 Specifically Inhibits Chronic Myelomonocytic Cell Growth
BION-1 is shown to inhibit the activity of one or all of IL-5, IL-3 & GM-CSF mediated effectors of leukaemic cells. In particualr BION-1 inhibits growth in vitro of chronic myelomonocytic cells (CMML), whereas a control antibody (1C1) does not (
Screening and Isolation of New Inhibitory Compounds
A large range of potential therapeutic compounds that might act as antagonists, or perhaps agonists of IL-3, GM-CSF and IL-5 individually or collectively, can be readily screened. The screening is initially to determine whether the binding of BION-1 or a fragment thereof to βc receptor or fragment is inhibited. The nature of these inhibitory compounds will not be limited, and the methods used for a binding assay can be any one of the many techniques known to those skilled in the art. Such methods may include affinity selection chromatography, ultrafiltration assays, the scintillation proximity assay, interfacial optical techniques, the quartz crystal microbalance, the jet ring cell, interferometric assays using porous silicon to immobilise the receptor. Reference to such techniques can be found in Woodbury et al 1999, which reference is incorporated herein in its entirety.
The range of therapeutic compounds may include peptides, oligonucleotides, or other small organic or inorganic molecules.
Deposit of Cell Line
The cell line BION-1 was deposited on the Apr. 29th, 1998 in the American Type Culture Collection (ATCC) at 101801 University Boulevard, Manassas, Va., United States of America and has been designated ATCC HB-12525.
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Fundamental Tax Changes Underpin Governor Wolf's Budget Proposal
Governor Wolf's 2015-16 Budget Proposal and the World Class Agenda
The Economy League assesses how Governor Wolf's recently-proposed state budget aligns with the priorities and strategies identified in the World Class Greater Philadelphia agenda.
How Immigrant Entrepreneurs are Driving Growth in Greater Philadelphia
Last month, the Fiscal Policy Institute and Americas Society/Council of the Americas released a new report on the impact that immigrant entrepreneurs – particularly, "Main Street" business owners – have on local economies. The report cites Philadelphia, along with Minneapolis-St. Paul and Nashville, as successful examples of how support systems for immigrant entrepreneurs can help foster overall economic growth.
Will New Jersey's Tank Run Dry?
Technology Transfer: Are We Making Progress?
Greater Philadelphia: A Brain Gain Engine
2015 Education Outlook: Issues to Watch
2015 may mark the beginning of some big changes in education in Greater Philadelphia -- many of which align with the priorities laid out in our shared World Class agenda. From key policy issues like fair funding to debates over curricula and teacher evaluations, here are a few key areas to watch this year:
Leaders Convene to Launch Greater Philadelphia Export Initiative
Last week, more than 70 business and civic leaders attended the final World Class Council forum of 2014 on "Spurring Regional Export Growth." At the event, the Economy League and the World Trade Center of Greater Philadelphia announced the launch of a new regional export initiative.
From Corruption to Collaboration in Chelsea, MA
The city of Chelsea, MA, has struggled for decades with high unemployment, crime and the legacy of political corruption. Last month, Greater Philadelphia Leadership Exchange participants traveled to Chelsea to learn how leaders are successfully working together toward long-term solutions to these often intractable issues.
A Few Lessons from Boston
"Eds and Meds": Anchoring Boston's Economy
In early October, more than 110 of Philadelphia's business, civic and government leaders traveled to Boston as part of the 2014 Greater Philadelphia Leadership Exchange. One of the key learnings from this trip was that in both Greater Boston and Greater Philadelphia, educational and medical anchor institutions serve as economic flotation devices, community partners, and catalysts for growth in their local communities and in their regions at large.
Development Without Displacement: What We Can Learn from Boston's Dudley Neighborhood
Earlier this month, the Economy League traveled north with more than 110 of Philadelphia's business, civic and government leaders to learn how Boston is tackling critical issues related to growth and opportunity. In this piece, we take a closer look at why the revitalization of Boston's Dudley neighborhood has served as a national model of resident-driven "development without displacement."
Beantown Meets Brotherly Love: Lessons from the 2014 Leadership Exchange
Executive Director Steve Wray offers reflections and insights from the 2014 Greater Philadelphia Leadership Exchange in Boston, MA.
Leaders Highlight Continued Need for Transportation Funding
With the passage of transportation funding legislation in Harrisburg last fall, southeastern Pennsylvania is slated to receive much-needed investment that will help address long-deferred maintenance needs and upgrade deteriorating systems. Achieving a World Class vision for our region's bridges, roads, and public transit, however, is going to require making the most of these and other resources.
As the Election Approaches, Pre-K for PA Gains Momentum
Education & Innovation in Massachusetts
Greater Boston and Massachusetts have a long history of innovation in education from the founding of the first publicly-funded school system in the US to creating grade levels and common standards that are the basis of our current education system to helping to build world class institutions of higher education. Today, the state and region continue to focus on systemic change and improvements along the education continuum.
Continuous Reinvention - Greater Boston's Knowledge Economy Edge
Historians point out that Greater Boston's economy has come back from the brink several times. In the post-World War II era, the region's economic success has been attributed to its foundation of science and technology strengths, educated workforce, and repeated ability to adapt to disruptions and changes in the knowledge economy. Whereas many regions, including Philadelphia, boast substantial eds and meds research institutions, Greater Boston's elite talent base and ability to reinvent its innovation economy have set it on a different path than other Rust Belt regions with similar research assets but economies that have faltered.
Giving More Kids Access to High-Quality Early Education
Driving Technology Transfer in Greater Philadelphia
In today's innovation economy, regions that are able to translate research discoveries into viable products and services are poised to capture significant growth. With Greater Philadelphia's world class academic and medical research institutions, converting promising ideas and technologies to the marketplace – a process known as technology transfer – is a crucial pathway for growing jobs and wealth in our region.

subset: commoncrawl


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Viewing the Pacific Ocean from Lāwa`i Kai, on the Hawaiian island of Kaua'i. World Ocean Day Photo Contest Submission by Dawn Forsythe.
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Liligo targets Spanish market with new release
The originally French travel search engine Liligo.com has launched a new version of its engine, releasing it also in Spain. There are new features integrated in the search engine which, amongst others, makes real time search through lights, hotels and car rentals possible.
Liligo says it offers "exclusive functionalities and additional features to further help travelers find the best deals to save money"
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A website that aims to make it easier for skiers to find detailed information about their chosen resort, such as which is the best apres ski bar, has been launched by a couple of British entrepreneurs.
Mountago.com is a user-generated digital directory which includes details about local businesses such as accommodation, ski hire, après ski bars and childcare.
Information is supplied by users and by local businesses, the site is free to use and an iPhone app will be added shortly.
"We want to get under the skin of a resort and provide a service where people can find everything they need," said Chris Purcell, one of the group of friends behind Mountago. The concept came from a ski trip last season when it proved a hassle to easily find the right place to go for a drink after skiing."
The site was launched with two French resorts, Morzine and Val d'Isere, but others are being added all the time, said the company.
All entries are verified before they go live and users can report any incorrect content.
Wednesday, January 23, 2013
Germanwings crew unfit to fly due to 'emotional distress'
Tunis massacre: Cruise passenger death toll rises again
Police make 'significant discovery' at co-pilot's flat
MH370 investigators examine towelette found on beach
UPDATED: Co-pilot of Germanwings flight 'intentionally started descent'
Hundreds of flight delays hit Heathrow
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text:
Country Christmas Tea
- Total Time:
- 2 hr 10 min
- Prep:
- 10 min
- Cook:
- 2 hr
- Yield:
- 10 servings
- Level:
- Easy
- Total Time:
- 2 hr 10 min
- Prep:
- 10 min
- Cook:
- 2 hr
- Yield:
- 10 servings
- Level:
- Easy
Pour 8 cups boiling water into a slow cooker. Add 16 chai tea bags; secure the lid with the tea bag tabs hanging outside and steep 10 minutes. Remove the tea bags. Stir in 1/2 cup each spiced rum and vanilla-flavored vodka and 2 cinnamon sticks. Cover and cook on low for 2 hours. Stir in 2 cups sweetened condensed milk; switch to the warm setting until ready to serve. Ladle into mugs and garnish with cinnamon sticks.
If you don't have a slow cooker, keep these drinks warm in a saucepan on the stovetop over low heat.
Photograph by Johnny Miller
Recipe courtesy Sandra Lee for Food Network Magazine
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Orange Tea with Honey
Recipe courtesy of Nancy Fuller
Low-Country Dogs
Recipe courtesy of Food Network Kitchen
Country Bread Stuffing with Goat Cheese, Kale and Bacon
Recipe courtesy of Bobby Flay


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Need help constructing a technobabble sentence
View Single Post
July 3 2013, 07:27 PM
Jonas Grumby
Vice Admiral
Location: Somewhere in the South Pacific
Re: Need help constructing a technobabble sentence
Morpheus 02 wrote:
The idea is to create a sentence that to non-scientists, sounds fairly scientific. But to scientists, it's utter bull-oney.
Does it have to be based on a sentence? There are plenty of things
in Trek that would make most scientists scoff.
Totally lifeless planets with oxygen atmospheres. Ancient, derelict spacecraft adrift in space for centuries, but with still perfectly functioning gravity, lighting, air and heat. Human-looking "aliens" everywhere.
Hell, just show 'em a shot of a spaceship traveling faster than light!
"There are two means of refuge from the miseries of life: music and cats." -
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|Research on Planning of Shenzhen Ping Hu Logistic Base
【Abstract】 The thesis of this paper is the planning of regional logistic base. The development of logistic base is still in the infant stage in China. The research on the planning of logistic base is of significant meanings in both theoretical and practical aspects for the regional industry restructuring and upgrade, and to facilitate the integration of regional economy and modernization of Chinese logistic industry. This paper solves the issues of how to planning and developing logistic base in a city through the research over the planning of Ping Hu Logistic Base. This paper consists of seven parts with main contents as follows:Part I – Environmental analysis, focusing on the study and analysis of Ping Hu Logistic Base's internal and external environment, including Shenzhen City and its peripheral area's industry structure and logistic infrastructure, and Shenzhen City's overall logistic planning, and the characteristics of Ping Hu Logistic Base.Part II – through the analysis of internal and external environment, the market opportunity, and the characteristics of Ping Hu Logistic Base, it is concluded that Ping Hu Logistic Base should be Shenzhen City's logistic center, and should be consisted of four core functional sections to target its functional positioning, including transport pivot, international transfer, regional distribution, and commodity trading.Part III – based on the positioning of Basic functions, the paper details the functional design of the four core sections, such as the requirement of land area. Lands are allocated to these four core sections in accordance with certain principles based on current land resources.Part IV – The design of information system, which will provide a platform for the projects in these four core sections and the enterprises in the Ping Hu Logistic Base to exchange information, thus it will improve the overall competitiveness of the <WP=5>Logistic Base.Part V – The functional positioning of the Ping Hu Logistic Base developer in different development stages.Part VI – financial analysis.Part VII – Investment promotion strategy, which decides the cooperation strategy, means, and methods for each of the four core sections based on the calculated direct and indirect returns in these sections.
|Ping Hu Logistic Base, planning, research,
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The former Harbor Gallery has reopened as (harbor), a space within a space, collaborating with our former neighbors, Regina Rex, to present an ongoing program of events and installations.
Please visit our new home:
221 Madison St. New York, NY, 10002
Hours: Thursday – Sunday 12:00 – 6:00 pm
We are pleased to announce a new iteration of our ongoing salon installation, featuring works by 15 different artists, each one selected by a different member of the Regina Rex crew.
Tatiana Berg
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OPENING RECEPTION// Sunday, January 11th, 2015, 6-8 pm


text:
Like I wrote - I first commented, than I watched the eclipse and now I came back to watch the video :)
Nonetheless I think you are right, of course we need "unicorn content" wich covers all goals, otherwise we get in trouble. We allways need value... Now I will watch ...
great video thumbnail for that topic, I usually use OG for Facebook titles, wich are completly different to the page title. Many people allways use the same title and tweet them (!) - i think that couldn't work well. But for CTR in SERPs, I take a look at them, when I rank and test some changes. But first the site needs to rank. A lot titles in SERPs here still look that way: Keyword | Keyword | Keyword - Brand/City - you can get a lot benefits and increasing CTR by writing a great title. It's pretty easy to write an SEO title like that, it's a lot hader to write a title in this clickbait-world. Hope your video would help, but first I watch the solar eclipse here (berlin)...
You can't be serious, what's with the page-layout-algorithm ... and how should that work? The more Ads the better you rank?
Transcript is the magic word - what should google now about a video. Content is King is just misunderstood. It is not enaugh to have a lot of "unique" Content, you'll need value :)
Content is King is often used, but knowone really thinks about it. Many people don't believe it, often it is misunderstood. And of course, there is no black and white, there is no one and only thing you have to do.
Now a bit content: Content is King is used so often and I think the first guy was that one: (http://web.archive.org/web/20010126005200/http://w... - amazing, in germany there was no internet available in most (even big) citys in 1996, but content was king.
Rank #1 for Magic Keyword
I had a client last year, he stopped with SEO because he didn't Rank #1 at his "Magic Keyword" - it was positon 5 from nowhere (bevor SEO - no ranking on that topic).
Organic Traffic increased 900% (!) in 6 month (a lot of technical and onpage failures) and he said: "SEO has brought me nothing". A lot more customers - but they didn't came from position one of his magic keyword.
Thats SEO live i guess. We talked a lot to him, he simply didn't want to understand or even listen, it was just 4 positions to bad!
SEO is all trick
You could say: SEO is all a cheap trick. We do our cheap tricks and see websites get well in ranking, right?
Google Answers
hard discussion in germany. How would it be, if google would answer here as much as it does in US? A massseosuicide would go on i think.
I think I heard everything of these Myth. An interesting thing is, we can hear (here in germany) that linkbuilding is dead and SEO is only Linkbuilding...
Great post, like to say as usually.
really - i mean, i only heard from that update, didn't thought that it is still possible... I am not that often in pigeon countries...
bing's keyword research tool tells me, that it is allways a good idea to use "google" as keyword... "what is bing" is the top '"what is" querry in bing.de - so many funny things...
great comment and they are shown in local packs, even in Germany :)
we dont have much, we dont know bing, but we have seo companies in (STILL) Local 7 packs - because we dont have a pigeon (thatswhy the local 7)
Ah ok, i would like to see facebook reviews here.. it is so easy to get reviews on facebook (again: in germany)
Ok I scored 80% but -
Q26 - I'll never see a review by facebook in germany, the others I even didn't know - so I think I am to european based to answer that :)
And Q17 - everything could be correct in germany :)
Last Question: as german I need to google what yahoo local is :)
Q38 - I also have to google what Bing is ...
(Google's actual marketshare in germany > 94% read it today and the most searched word in bing: Google)
I like this Quiz - thanks for that!so there I was wrong, but its Ok... btw - I have to admit, I worked with google translator a lot.
great metapher, but I can understand the webmasters. They allways read about links and how important they are for ranking. They worked and payed a lot for all these links and wont lose one. There are not so many (shown) examples of people wich get back on top in disavow 50% (or more) of there links.
But they exist...
hire a web developer who knows to include all of these functions rather than to hire both a web developer and an SEO
Simply not possible to hire a web developer who is perfect in SEO (even if you only think technical SEO) - I guess it's easier to hire an SEO who knows everything you need for your web development.
It's good if a web developer knows a lot about SEO, but I would never miss the knowledge of an SEO pro for sure. Things a developer may never think about can impact ranking.
But that's my opinion as SEO :)
I really think each of this Jobs - PR, Web Design, Web Development, SEO are so special, that it is always a great choice to have at least one specialist of each area in your team. PR or Designer trained by an SEO? For really great SEO work you need passion, I work in a web design agency, maybe one of our designers is interested in SEO. Same at the DEV Team. But both don't have enaugh time (not only to learn SEO) to see the SEO affect of some things they do. To realize that it has or could have (may be in future) an impact. They just have to do the job they are paid for.
maybe the haircut is a little older, but the style looks very differnt in this video, than in the video before :)
"but Google recognizes I'm searching from Portland" and that is what many of my clients don't understand or believe(!) - ok it is not portland, its more Berlin or another german city.
I even can't tell them, that a restaurant 60miles away from Berlin would never be #1 on "italian restaurant berlin" because they aren't from berlin. Local SEO is hard in germany ;)
Wich we here would have a tool lice MOZ Local.
Great that MOZ Analytics delivers local rankings now - and again I wich we would have such a tool in germany
wow - thanks for that Gianluca Fiorelli.
Never thought much deeper about the difference between "Value" and "Quality"...
I thought that #2 "We will see Google test search results with no external, organic listings" couldn't be possible and commented that last year and I glad that we didn't saw that and (like you) hope we will never see it.
I would say:
In Europe Amazon will become a harder opponent for google in transactional queries (if we search somtehing to buy, we often search at amazon, more often than with google not only in my case, mostly caused by bad google shopping experience and much more)
A pretty big problem, for ,many client sites I don't have the autority to take a look on these factors. That they do matter (may be a lot more than we think) is clearly visible, when I watch the pages, where I have the autority and work on these factors. They don't struggle that much or move up and down. I have to work less for them... Have a nice 2015 everyone!
I think it is pretty hard to get a good timeline.
I usually do it in that way, milestones, some goals we want to reach in time, but it is pretty hard to find the time you will need. Thats worth a WBF by itsselfe. How to create a realistic SEO Timeline
so many clients often say: I know that it would take a while. I know that it will need time.
But what is a a while or time? 3 Month, 6 Month, a year?
It is pretty hard to say what is really possible in 3, 6 or 12 month. Talk a lot to the clients, helps a lot. Tell them what you do and what your goals are in the next few month. After that analyse it with them and search some new goals for the next month. Never stop to communicate with them... Thats what I see.
@Tony Dimmock When something is impossible, it is easy to say no - but what if you like to say "maybe" ?
I think he wasn't thinkin about: smashing an infographic (what a really good linkbait was) and tell us how it would be better, he just wanted to say: stop thinking there are exactly, straight 200 factors, stop beleiving start knowing.
I think he said it really clearly here: my intention is to make clear—especially to you, young SEOs—that nothing good can come of your taking these lists at their word
... Gianluca Fiorelli as allways a thumb up collector. You are right and I just want to give a thumb up. But as allways I can tell you a little inside view into german SEO.
Like AngelaMerkel told us a while ago - internet is #neuland wich means it is new for us - thats a bit funny in 2014. But SEO seems to be pretty #newland for us - because all these SEO tactics we would see as: old and not usefull is often used. Bought links, bad directorys, just get the link, comment spam... I know several sites with a link profile like this. And AB Tests? Her is what we do in general:
In general we don't do A-B Tests. Yeah right, there are SEOs maybe more than a handful wich do AB Tests and several webmasters (wich call themselfe SEOs) wich don't use it at all. They still buy Links on ebay and thats all. #Shame but we are #Weltmeister in other games :-)
I get 20 mails a month from "SEOs" wich want to sell Links to me, or buy links from me. In the case of selling - I ask what they do, what these Links would give to me - what do there Tests say about it? And they say: We don't do Tests - Links help - no matter what link.. trust me...
I think we are talking about the internet, the internet is Links - links from one page to another. Isn't that how it works? You write something about a page and link to it - followed, because that is the internet.
Its unnatural to link out only nofollow.
I use nofollow only for Blog Comments (name Link) and when I write about a page I want to dissociate.
Great, its allways a question how porn sites make their SEO, but how do they get there social followers? Not one person I know is following a porn site ;)
I think that is a great question .. I mean who of you is following them via Twitter?
How they do the job is great and you can see it is more than directorys to win that game - it really doesn't matter what your topic is - great post with nice infos I didn't know (the justin beaver tweet)...
You can change the Text in Chrome and Firefox (I think also without firebug) and with the latest internet explorer its possible to. I only write it, because you seem to like MS products :)
Never thought about ms paint, but I still use Office and the Snipping tool - like you said, learned it and did it over years make you pretty fast :-) And the snipping tool ist just a good tool, sometimes the easy and small tools are the best.
I really hope that SEO would never be so "easy". Crawleability and great content... But o.c. great content isn't easy written... SEO touches everything and many people didn't even check it.
So many great linkbaits shared via social networks and noone knows that there was a pretty great SEO team behind it.
Great WBF - special people wich mention you, but don't link. I don't know if its a german problem, many people dont link to a site. Some are linking to the wrong root domain (www / non www f.ex.). I got so many links in that way!
But last week I was ill - so ill that I not even take a look at my Mails (yes possible / thatsway I watch the WBF today xD ) - When I looked at my mailaccount (I have special mail accounts for alerts) I got so many Mails... ouch!
dont get me wrong, I don't want to say Analytics is the best tool. I am one special case and I would say If you can try all tools ;)
Piwik is to slow for the Data Sets I need - so it's not Piwik in real, its the server where its installed. So they client hosts are the Problem. When I need Data for 360 Days I can go out, drink a coffee and came back and see - an Error, again. Anoying. I know thats not really a Piwik problem, but thats my case.
plus I need to check and install Updates - brr
I used Piwik for my private sites, fast and without errors - and my Hoster updates it for me. In that case it works great. But my clients sometimes have bad providers wich they dont want to leave...
I tried McDonalds and Burger King and now I decided for McD :)
Or I gave Piwik a try and I am not a Fan of this analytics tool for several reasons. I can understand what you mean, but I am not that sure, thats it is worth the hours of work to try other tools out, as long as I can get all that I want very fast with a tool I allready have.
Maybe I would work several hours just to say at the and - ok I allready used the best option. I tried more tools than Piwik and Google Analytics - atm Google is the best option of all tried tools. Maybe there is one wich fits better to me, I actually do not know :(
I work with analytics and piwik, and when I have to chose one it is analytics.
Of course it's not that easy to see inside and get great view inside to your visitors, but didn't you said "easy" is not a think that should us let decide about an analytics tool?
There is a point you can see here allready - the community, what google analytics has, here is the first person asking about adobe digital analytics. When I have a question how to get some usefull data out of analytics, I could find several posts with right answer.
I think he means meta tags like that one:
<meta itemprop="" content="">
in germany often used to fake ratings
it is possible to highlight things, wich are not visible for Users - I never understood why that is possible.
who talks about spamming?
I mean - it is possible that a webmaster is just linking all his sites together and he just didn't knows it better. For him it's good to know what not to do.
And everything what shows how google ranks websites has to be interesting for SEOs and Online Marketers.
( the greatest content with no link = invisible for google ;) )
I know a Domainhoster who tested it.
Fact was: many Linking Domains (>1000) with KW in anchor text, but also the brand (25% on the same c-block, only 4 c-blocks so: 250 linking Domains in each block). Additional ~1000 Links from Domains with a normal Linkprofile :-)
But, the site wich got the link was (like I told above) a website hoster. maybe google noticed that, and it was a hoster bonus... dont know that? Maybe the other 1000 Links brought the value and the rest was for nothing. He spoke from a ranking win - don't no more about that. Maybe there wasn't really one - I am not his SEO just a friend ;)
Amazon is just making a pretty good job. Its no deal. I read somewhere that google penalized ebay for stopping adwords - thats freaky and absurd.
I will take a look on ebay.de asap - maybe its the same here.
~30% visibility loss for ebay.de
more about ebay.com loss: http://refugeeks.com/deconstructing-ebay-coms-organic-loss-using-semrush/
sorry now I can see DrPete allready posted the link...
I think it would come a pinguin soon - I also wait for it. Have a client domain where Pos 1 to 8 in google SERPS definitly won't survive a penguin...
OMG really?? Huh...
Ok back to topic, like the idea of socials, for some clients I dont have any other chance but the socials are a great help. Special to watch who is following the big onces and which other sites do they follow, what they like, ... all these stuff is really helpful.
Have a great friday 13th
when you can manage to target the consumers earlier, when answer there questions - and your website is pretty unique or your logo is, you can see that people start trusting more and more in your company and your knowledge about that topic they are searching for. You get a trust boost powerd by google. I see that pretty often, for many clients I start to target the topics as early as possible. Great advice
great - if something is not broke - dont fix it
and if you have rankings - never change a winning team
^^ depands on the costs, but its a great advice i think. I fixed many titles last weeks - but for sites wich didn't ranked well or wich truly needed a new one and I didn't made it till now :)
Great WBF
it is what we are doing. But when you do something like that you start 301 the equal an old posts. new design - make it responsive ... thats hard work now.
@Michael - I know thats why that 301 was one of my first changes when I started - but its a lot work
whats wrong with the lights? its not that great lighted as usually and I always looked at one of the two shadows!
other location??
subdomain blog ... äh, I am actually working on a blog - thousends of posts - on a subdomain... how much work it is to get it corectly in a subfolder - make the 301, than (when u work on it you can truly change the design and with that, the cms to) so much work, because one guy didn't thought about something a few years ago.
Hope you all never have to do that. Because, the blog earns links, socials, content... but on a subdomain :(
great WBF - nice Weekend everybody!
?? hmm - do you mean the background of his pic and yours - and that the post is cool because the pic backgroung is? Green stands for calm, freshness and health?!
Hey Rand also has a new green background..
Try'd reddit - its not a great deal in germany... Got allmost us / englisch speaking traffic, works well for english language sites, so for me, it works for the global pages. I never registered that it is possible to make ads :)
Thats how interesting is reddit for am german ;)
I love the idea - but it doesn't work ... now I am sad...
it's a mean trap, easy to overlook when you watch the site in the browser (you are not used to look above the header) and a trap wich you don't think about when you insert the description.
Even Roger is a human :)
lol right - i just saw the xyz> and thought there must be an lost "<" ...
Its Friday so don't be to hard to me.
So the first 15 FB shares had no description ;)
ah ok - directly next to the video - watch you look for 10 minutes is a big red banner "start my free trial" - may be that is more powerfull than one sentence in the video?!!
right, strategy and a long way and much time for unique and great content creation. Not buy 20 Articles and get one free. That couldn't work.
comon - I am not working for MOZ :)
I just mentioned it because rand did - 7 visits till free trial. Upps I did it again...
back from easter holiday by the way? x)
Content Marketing is tough and needs a strategy. Thats the good and bad thing. If it would be easy, it would end like comment spam - everydone would do it, the effect wouldn't be that great.
Content Marketing main purpose is thought leadership - thats what I think to! Great Comment.
Have a nice WE everybody!
It is interesting how MOZ measures it that, and what is the success - hard to measure - is only the conversation success? Is it success when someone shares or likes content? Or when he Links, comes back and starting good conversations in Comments. Or when many other Blogs arround the world translate that content and use it in there blog - hopefully with a link to the original one.
It takes 7 visits until a user will take part of a free trial? I think thats not that bad :)
I think one of the interesting parts is: dont give up way to early
That is what a lot of people doing, and dont envolving SEO , also is a big fault I see a thousend times.
I also see backlink profiles with 70% exact match anchor.
Many of them external - follow - in the footer of a page and the rest are questionable bookmarks and directories.
And thats no fun, they rank well to the anchor of course - but how long....
Its "OK" when your brand is just not important for, but I do not want to have such a Backlink profile - not for me or my clients...


text:
Witches Fly There explores magical healing and worldview in the East African country Zambia. Woven into modern Africa is a mythical dimension, which it is hard for us to perceive as anything but superstition.
This film leaves the prejudices behind and takes the viewer head first into the living mythologies of modern Africa.
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Products & ServicesSee also: Categories | Featured Products | Parts by Number | Technical Articles | More Information
Supplier: Versa-Matic Pump Co.
Description: . Features use FDA-approved 4059FG-grade Hytrel® rubberized plastic are excellent in abrasive product transfer applications are also available for Wilden® pumps
Description: Diaphragm Replacement Kit, Material of Construction Plastic, Rubber And Stainless Steel, Spring Stainless Steel, Application For Replacing A Diaphragm In A 3/4 In Or 1 In CP Series In-Line Valve
Supplier: Clark Solutions
Description: Model B502 Plastic Air Operated Diaphragm Pump Flow Rates to 650 LPM (89.8 GPM), Pressure to 70 Meters (99.7 PSI) DESCRIPTION B502 plastic diaphragm pumps are characterized by exceptional performance, power and strength, making them ideal for pumping liquids with very high apparent viscosity up
- Maximum Discharge Flow: 10296 GPH
- Maximum Discharge Pressure: 99.7 psi
- Inlet Size: 2 inch
- Discharge Size: 2 inch
Description: handwheel makes it easy to open or close the valve, and a beacon-type indicator provides highly visible position indication at the top of the valve's rising stem. Rated to 150 PSI @ 70°F, non-shock. Additional diaphragms and materials available, contact us for quote.
- Valve Size: 1.5 inch
- Connection: Union
- Material of Construction: CPVC
Supplier: Hayward Flow Control
Description: , CPVC, PP, and PVDF Position Indicator Sure-Grip Handwheel Choice of FPM, EPDM, or PTFE Diaphragms
- Material of Construction: PVC, Polypropylene (PP)
- Valve Type: Diaphragm Valves
- Valve Size: 0.5000 to 10 inch
- Pressure Rating: 75 to 150 psi
Supplier: Valcor Engineering Corporation
Description: The SV73 series is a 3-way, balanced poppet isolated diaphragm solenoid valve. These valves are designed for the control of corrosive fluids by isolating all the metallic components from the operating media. This series employs two rolling diaphragms, acting both as isolating barriers
- Valve Type: Solenoid Valves
- Valve Size: 0.7500 inch
- Pressure Rating: 145 psi
- Number of Ports/Ways: 3
Description: Replacement Sleeve, Plastic
- Market / Industry: General Industrial
Description: capability in either flow direction, and having diaphragms being essentially made of elastomeric or plastic material or combinations thereof. This Standard Practice defines the following categories of valves: a) Class 125 or 150 Valves; b) Standard Valves; c) Valves for Special (Low Pressure
Supplier: Applied Industrial Technologies
Description: 1 in. (25.4 mm) ANSI end-porting High flow - 40 gpm (151 lpm) in a compact, portable size Fluid pressure to 120 psi (8.4 bar, 0.84 MPa) External controller for remote control access All Graco diaphragm pumps offer flow rates up to 25% greater than many competitive pumps with PTFE diaphragms
- Maximum Discharge Flow: 2400 GPH
- Inlet Size: 1 inch
- Discharge Size: 1 inch
- Media Temperature: 180 F
Supplier: Asahi/America, Inc.
Description: in applications such as slurries or suspended particles Bonnet seals to protect internals from corrosive environments Built-in travel stop to prevent diaphragm from being over-tightened Indicator at the top for valve position All valves with Teflon diaphragms come with PVDF gas barrier, which protects
- Material of Construction: PVC, Polypropylene (PP), PVDF
- Valve Type: Diaphragm Valves
- Valve Size: 10 inch
- Pressure Rating: 65 psi
Description: Diaphragm Pump, Motor Driven, Inlet/Outlet 3/8 In, Max. Flow 2.2 GPM, 24VDC, Max. Operating PSI 60, Polypropylene Body Material, Buna Diaphragm Material, Polypro Ball Material, Buna Check Seat Material, Max. Temp. 140 Degrees F, Length 7-15/16 In, Width 4 In, Height 3-5/8 In
- Market Segment: General Industrial
Description: Intake/Discharge: 1/4" FPT and 1/2" MPT. Chamber Material: Polypropylene. Diaphragm Material: PTFE. Max Temp 150°F. Max Solid Dia.: 1/32" Overall size: 7-13/16" x 7" x 5-1/2"
Supplier: Clark Solutions
Description: is a three-way configuration. Model WTB-3R has molded PPS bodies and Model WTB-3K has molded PEEK bodies. The valves have PTFE isolation diaphragms and a Perfluor or FPM soft valve seat seal.
- Material of Construction: Other
- Valve Type: Diaphragm Valves, Directional Valves, Solenoid Valves
- Valve Size: 0.2500 to 6 inch
- Pressure Rating: -26 to 29 psi
Description: The SV-10 Series solenoid valves for corrosive applications use only plastic or elastomeric materials: PVC for the body, Buna-N, EPDM, and FKM for isolating and main diaphragms, and a fully epoxy-encapsulated pilot solenoid actuator. The pilot actuator uses the pivoted armature design, which
- Material of Construction: PVC
- Valve Type: Solenoid Valves
- Valve Size: 0.5000 to 1.5 inch
- Pressure Rating: 85 psi
Description: Isolate and protect gauges and other instruments from contracting the process fluids. Gauge Guard has a FKM diaphragm to separate the process media from the upper chamber, which is filled with hydraulic fluid. The force of the system pressure exerted on the diaphragm displaces the fluid in the upper
- Diaphragm Material: Other
Supplier: Cole-Parmer
Description: • Designed for use in corrosive environments • Multi-turn handle gives you precise control • Position indicator shows exact location of valve • Self draining allows for little dead volume in valve • PVC body and EPDM diaphragm • Tru-union connections
- Material of Construction: PVC
- Valve Type: Diaphragm Valves
- Pressure Rating: 225 psi
Description: The FPUD1000 Series are positive displacement metering pumps. The FPUD1000 Series is designed to inject chemicals into piping systems and is capable of injecting against a high system pressure up to 150 psi (10.4 bar). All models are equipped with a front mounted mechanical flow rate adjustment
- Maximum Discharge Flow: 1.59 to 15 GPH
- Maximum Discharge Pressure: 150 psi
- Inlet Size: 0.3750 inch
- Discharge Size: 0.3750 inch
Supplier: Radwell International, Inc.
Description: Are you looking for the best website to buy GRACO D52977? Then you have found exactly the industrial MRO outlet you need right here! At PLCCenter.com our #1 goal is the satisfaction of our customers - we strive to develop market-leading products and services that are offered to you at an exceptional
- Maximum Discharge Flow: 3600 GPH
Description: Pumps are air-driven, double-diaphragm pumps. The simple design and operation offer many advantages over other types of pumps. Ideal for OEM applications and offers multiple mounting positions. Great for car wash chemicals, wash solutions, dispensing of pigments, inks, paints, additives and
- Media: Combustible, Gasoline / Diesel Fuel, Liquids
- Power Source: Air / Pneumatic
- Market / Industry: General Industrial
- Double Diaphragm Pumps: Yes
Supplier: Saint-Gobain Plastic Films and Tapes
Description: FEP Type FS FEP Fluoropolymer Film FS grade film is a translucent film made frm high molecular weight resin for applications where high flex life and stress-crack-resistant performance are required, such as tank linings and diaphragms.
- Chemical / Polymer System Type: Fluoropolymer (PTFE / PVDF)
- Thermoplastic: Yes
- Industry: Aerospace, Electric Power, Medical / Food (FDA, USDA), General Industrial
Supplier: Savino Barbera Srl
Description: Simple and quick maintenance and with easy-to-replace components, diaphragm pumps BX 100 are fed with non-lubricated air and have an adjustable flow rate and head. Their explosion-proof properties have been certified for the ATEX version according to Directive 94/9/EC ATEX Equipment. Other technical
- Maximum Discharge Flow: 2376 GPH
- Inlet Size: 1 inch
- Discharge Size: 1 inch
- Power Source: Air / Pneumatic
Description: Diaphragm Valves, Pipe Size 1 In, Material of Construction CPVC, 150 PSI Max. Pressure, Temp. Range 40-190 F, Length 5.5 In, Seal Material EPDM, Diaphragm EPDM, Manual Operation, Cv Flow Factor 13, Connection Threaded
- Material of Construction: CPVC
- Valve Type: Diaphragm Valves
- Valve Size: 1 inch
- Pressure Rating: 150 psi
Supplier: Radwell International, Inc.
Description: Finding an industrial MRO supplier with YAMADA PUMP DP20-F in stock isn't always easy - you need a experienced, global company with a high level of both knowledge and service. At PLCCenter.com, we stock New and Surplus Industrial MRO products for business and consumers all over the globe. In order
- Primary Material of Construction: Plastic
Supplier: GRI Pumps (A Gorman-Rupp Company)
Description: Piston diaphragm pumps are positive displacement pumps designed to economically transfer small volumes of fluid at low discharge pressures. The lightweight, compact pumps are self-priming, corrosion resistant and can handle viscous fluids. These features make the pumps ideal for high volume copy
- Maximum Discharge Flow: 0.2376 GPH
- Maximum Discharge Pressure: 2.6 psi
- Inlet Size: 0.1875 inch
- Discharge Size: 0.1875 inch
Supplier: Federal Equipment Company
Description: Find the right used industrial pumps for your application by browsing their inventory. At Federal Equipment Company, they're committed to providing their customers with low cost, high-quality used industrial equipment. Their selection of used piston pumps includes: • Carbon Steel Pumps •
Supplier: Ark-Plas® Products, Inc.
Description: . Check Valve diaphragm standard choices include Neoprene, Silicone, and Viton. Diaphragms should be matched closely to the body material by likeness in physical and chemical properties. This helps ensure that the best possible performance is achieved from Ark-Plas® Check Valves.
- Material of Construction: Polypropylene (PP)
- Valve Type: Check Valves
- Valve Size: 0.1875 inch
- Pressure Rating: 1 to 150 psi
Supplier: Equilibar, LLC.
Description: Precision Back Pressure Regulator For liquid, gas & mixed phase processes SS316 Rugged Bar Stock Design Unique Chatter Suppression Feature in liquid only applications High Precision and sensitivity PTFE, stainless steel, and Hastelloy diaphragms available The NL series back pressure
- Material of Construction: Acetal, PVC, CPVC, PTFE - Teflon®, PVDF
- Valve Size: 1 to 4 inch
- Pressure Rating: 0.0 to 500 psi
- Number of Ports/Ways: 2
Description: The FPUDT1500 Series injector pumps feature a heavy-duty cartridge valve pumphead made of injection-molded polypropylene. The pump includes double-sealed PVDF cartridge valve fittings, FKM O-ring seals, ceramic ball checks, Hastelloy C valve springs, and a PTFE/EPDM diaphragm. Units with the
- Maximum Discharge Flow: 0.3487 to 4.99 GPH
- Maximum Discharge Pressure: 70 to 125 psi
- Inlet Size: 0.2500 inch
- Discharge Size: 0.2500 inch
Description: GPD: 22.8, GPH: 0.95, ML/Min: 60, Max PSI: 115, Seals: Viton, Diaphragm: PTFE, Discharge Tube: 3/8" PE
- Pump Type: Positive Displacement Pumps
Diaphragm Pumps - 400-1941 - Air Cadet Vacuum/Pressure Pump, OEM, 0.65 CFM, 20" HG, 115 VAC -- GO-07530-85Supplier: Cole-Parmer
Description: • Unique cavity configuration minimizes dead space and air entrapment • All-plastic, convoluted diaphragm design handles many vacuum/pressure applications and reduces stress, wear, and heat build-up. • Pumps are available with standard 115 or 230 VAC motor; or portable 12 VDC motor
- Maximum Discharge Pressure: 18 psi
- Maximum Discharge Flow: 269 GPH
- Power Source: AC Powered
Supplier: International Polymer Solutions Inc.
Description: Our Inline Diaphragm Valves (IDF) features an all wetted PTFE flow path with a diaphragm design for ultra pure service. Common applications include DI Water, Acid Drain, and Source Chemistry flow shutoff. The IDF valve is offered in a two-way closed configuration. All PTFE wetted flow path. Long
- Material of Construction: PTFE - Teflon®
- Valve Type: Diaphragm Valves
- Valve Size: 0.5000 to 0.7500 inch
- Pressure Rating: 80 psi
Supplier: G.I.E. Inc.
Description: GIE's broad line of diaphragm valves is widely applied throughout the chemical, food, paper, steel, mining, pharmaceutical, and other process industries wherever positive shut off, non-contamination, or ease of maintenance is required. Available with a wide range of manual and automatic actuators,
- Material of Construction: PVC, Polypropylene (PP), Other
- Valve Type: Diaphragm Valves, Shut Off Valves
- Valve Size: 8 inch
- Pressure Rating: 200 psi
Description: Diaphragm pumps handle water and water-like liquids and sludge. Self priming with 12 ft. suction lift and 12 ft. head. 10 gpm model (9 strokes/gal.); 12 gpm model (7 strokes/gal.); 25 gpm model (3 strokes/gal). Reinforced Buna-N diaphragm and flapper valve. Tough anodized aluminum handle. Foot
- Maximum Discharge Flow: 720 GPH
- Power Source: Manual
- Market / Industry: General Industrial
- Inlet Size: 1 inch
Description: Normally closed valveCreates a vacuum when switching from open back to closed position to suck back a predetermined amount of liquidAdjustment gives excellent repeatability on the suckback volume
- Material of Construction: Other
- Valve Type: Diaphragm Valves
- Valve Size: 0.2500 to 0.5000 inch
- Pressure Rating: 60 psi
Supplier: DeWAL Industries, Inc.
Description: transmission cable, computer wire, microwave, process filtration, battery and fuel cell separators and instrument wiring. It is also usable in the chemical process industry for gaskets, expansion joints, valve seals and diaphragms.
- Chemical / Polymer System Type: Fluoropolymer (PTFE / PVDF)
- Filler Material: Unfilled
- Material Type / Grade: Dielectric / Electrically Insulating
- Industry: Energy Conversion (Battery/Fuel Cell/PV), General Industrial
Find Suppliers by Category Top
Featured Products Top
Hayward Flow Control
Manual Diaphragm Valves
GENERAL DESCRIPTION. Overview. Hayward all-plastic diaphragm valves are engineered to provide superior handling of difficult media such as corrosive fluids, abrasives and slurries. They can also be used for high purity and sanitary applications. Manual or Automated. Available in manual or actuated (pneumatic or electric) models, the valves can be used for on/off and throttling service, feature a multi-turn design for precise control, and are self-draining on one side so that little to no dead... (read more)
Browse Diaphragm Valves Datasheets for Hayward Flow Control
Girard Rubber Corporation
Diaphragms - Elastomers
Product Families - Elastomers. •. Gaskets - "O " Rings - Seals. •. Pump Impellers. •. Diaphragms. •. Molded/Laid-Up Tubes & Hoses. •. Shock Mounts. •. PTT/Volume/Squelch/Function Buttons and Knobs. •. Keypads and similar decorated items. . •. Silk-screened & hard-coated. •. Laser-Etched. •. Back-filled. •. Die-cut Rubber/Plastic/Laminate Gaskets, Seals, Pads with and without Pressure-Sensitive Adhesive Backing... (read more)
Browse Rubber and Elastomer Molding Services Datasheets for Girard Rubber Corporation
G.I.E. Inc.
Diaphragms for use in Saunders ®
Flows in either direction. • Isolation of bonnet from fluid. • Easily cleaned with variety of pigs. • Simple in-line maintenance. • Consistently leaktight, even with solids in the line. Dimensions. SIZE INCHES 1/2" 1" 1/2" 2" 2 1/2" 3" 4" 6" 8" 10" 12". A UNLINED 4 5 1/2 6 1/2 7 1/2 8 1/2 10 12 1/2 16 20 1/2 25 29 1/2. RUBBER LINED 4 1/4 5 3/4 6 3/4 7 3/4 8 3/4 10 1/4 12 3/4 16 3/8 20 3/4 25 3/8 29 7/8. PLASTIC LINED 4 1/4 5 3/4 6 3/4 7 3/4 8 3/4 10 1/4 12 3/4 16 3/8... (read more)
Browse Diaphragm Valves Datasheets for G.I.E. Inc.
G.I.E. Inc.
G.I.E. Straightway Diaphragm Valves...
Flows in either direction. • Isolation of bonnet from fluid. • Easily cleaned with variety of pigs. • Simple in-line maintenance. • Consistently leaktight, even with. solids in the line. Dimensions. SIZE INCHES 1/2" 1" 1/2" 2" 2 1/2" 3" 4" 6" 8" 10" 12". A UNLINED 4 5 1/2 6 1/2 7 1/2 8 1/2 10 12 1/2 16 20 1/2 25 29 1/2. RUBBER LINED 4 1/4 5 3/4 6 3/4 7 3/4 8 3/4 10 1/4 12 3/4 16 3/8 20 3/4 25 3/8 29 7/8. PLASTIC LINED 4 1/4 5 3/4 6 3/4 7 3/4 8 3/4 10 1/4 12 3/4 16 3/8 20 3/4... (read more)
Browse Diaphragm Valves Datasheets for G.I.E. Inc.
Hygenic Corporation (The)
Rubber Die Cutting: Plastic Sheet or Film
RubberDieCutting: Hygenic can provide custom die cutting from any of our flat sheeting and film materials – HYTONE ™ Natural Rubber Latex Sheeting, HYTONE ™ Natural Rubber Latex Film, HYSYNAL ® Synthetic Rubber Sheeting, and HYPERFORM ™ Elastomeric Sheeting and Film. Our die cutting services are often used to make medical industry products. Rubber sheeting and film can be used for die cut gaskets, seals, diaphragms, and trocars. All die cutting services are provided... (read more)
Browse Die Cutting Services Datasheets for Hygenic Corporation (The)
Plast-O-Matic Valves, Inc.
Diaphragm Seals Reduce Instrument Failures!
to Series GGMU users. CAD Files. Online Pressure Drop Calculator -- Allows you to easily determine the probable pressure drop across a valve. Note: For comparison and illustrative purposes only; not to be relied on for system design. Plastic Valves and Controls. Quality Engineered for Corrosive and Ultra-Pure Liquids. Solving pressure & flow control problems for a variety of applications. Founded in 1967, Plast-O-Matic Valves strives to continually advance the technology of liquid pressure... (read more)
Browse Diaphragm Seals Datasheets for Plast-O-Matic Valves, Inc.
GF Piping Systems
Diaphragm Valve-Improved Flow, Reduced Energy Use
water treatment, cooling and control. GF Piping Systems supplies a full range of plastic pipe, fittings, tubing, valves, actuators, rotameters, fusion machines, secondary containment, tank linings, heat exchangers, custom products, and sensors and instrumentation for industrial process control. For further information, please contact GF Piping Systems, 2882 Dow Ave., Tustin, CA 92780-7258; Toll Free (800) 854-4090, Fax (714) 731-6923; e-mail: firstname.lastname@example.com;. Web: www.gfpiping.com... (read more)
Browse Diaphragm Valves Datasheets for GF Piping Systems
U.S. Plastic Corporation
ARO® Dosing & Transfer Double Diaphragm Pump
. Polypropylene seal material. PTFE ball & diaphragm material. ... (read more)
Browse Dosing Pumps Datasheets for U.S. Plastic Corporation
Plast-O-Matic Valves, Inc.
Diaphragm Check Valves are Superior to Ball Checks
PLASTIC CHECK VALVES. True Blue ® PVC, CPVC, PVDF, PP & PTFE Check Valves help you stop unwanted reverse flow with an automatic bubble-tight seal. • Series CKM & CK Diaphragm operated; self-sealing and normally-closed. • Series CKS PFA encapsulated spring designs; self-sealing and normally-closed. • Series CKD Compact design for low pressure applications; not self-sealing but has lower cracking pressure. Diaphragm Check Valves are Superior to "ball checks... (read more)
Browse Plastic Valves Datasheets for Plast-O-Matic Valves, Inc.
U.S. Plastic Corporation
Plast-O-Matic VBM & VBS Diaphragm Vacuum Breakers
Protect Against Hazards, Damage and Financial Losses Caused by Vacuum!. Designed to protect enclosed tanks from collapse or structural damage during draining. Eliminates siphoning of dangerous fluids and prevents vacuum which can cause damage to sensitive instruments and filters. Insure against replacement of damaged expensive equipment and avoid critical system downtime. Patented diaphragm design assures dependable, repetitive, bubbletight sealing in VBM; PFA encapsulated spring and special... (read more)
Browse Industrial Valves Datasheets for U.S. Plastic Corporation
Parts by Number Top
|Part #
|Product Category
|Pneumatics, Pneumatic Valve
|Machine Parts, Gasket
|National Microchip
|Richco Plastic Co
|Not Provided
|National Microchip
|Richco Plastic Co
|Not Provided
|National Microchip
|Richco Plastic Co
|Not Provided
Conduct Research Top
Plastic AODD Pumps for Manufacture of Solar Cells
When considering solar or photovoltaic cell manufacture, there is really only one choice for your pumping needs - plastic air-operated double-diaphragm pumps. Due to their broad range of applications based on the different types of liquids that need to be handled, plastic AODD pumps can be used
The Sky's The Limit- Plastic AODD Pumps in the Manufacture of Solar Cells (.pdf)
involved in the production of solar and photovoltaic cells, there is a pumping technology available that offers the design, materials of construction, energy consciousness and operational efficiencies needed for this critical application. They are called Plastic Air-Operated Double-Diaphragm (AODD) Pumps
Take Control of Your Operations - Proper Air-Control Systems Increase Efficiency and Reliability of Pneumatic Diaphragm Pumps (.pdf)
Plastic pneumatic diaphragm pumps are used widely in chemical, semiconductor and pharmaceutical applications, as well as other industries that require sanitary operating atmospheres. These types of pumps are ideal for these applications because of their almost universal chemical resistance-since
Tests Show That Solid-Body Plastic Pumps Are The Best Choice For Oscillating Applications (.pdf)
the machine is started and then remains constant, the components of an oscillating machine-such as diaphragm or piston pumps-are subjected with each single discharge stroke to continuous alternating stress between the maximum delivery pressure and the maximum vacuum in suction operation.
Medical Device Link .
of rubber components bonded to aluminum, brass, steel, Teflon, and plastic. The company also supplies a range of custom formulated and compounded elastomers to meet correct functional, environmental, and color requirements of the bonded assembly. Many industry requirements can be met with the custom
Medical Device Link .
A membrane micropump offered by a developer of microfluidics systems for medical technology, pharmaceutical research, and other applications uses piezoelectric actuation in conjunction with flap valves to rectify the flow. Liquids and gases can be pumped by the long-lasting all-plastic mp5
Medical Device Link .
or less. However, achieving high accuracy on very small volumes of aggressive media over as many as 30 million cycles requires more than putting an isolating diaphragm on a standard solenoid valve and machining a plastic body. A number of proven suppliers are acquiring extensive experience
Case Study: Machining - VMC for Real-World Moldmaking and Milling - 04/08
SHM is a moldmaking business within a 2,500-square foot facility that specializes in compression, transfer and injection molds for the rubber industry. According to Jack Latragna, owner of SHM, rubber molds are different than plastic molds in that they can be undercut and don 't need slides, so
More Information Top
Application of a plastic diaphragm for a silicon micropump
Application of a plastic diaphragm for a silicon micropump .
Numerical simulation of ground water protection works for an industrial waste dump
EX PIT Plastic diaphragm .
Engineering Geology for Society and Territory - Volume 2
149 Use of Plastic Diaphragms to Improve the Resistance of River Embankments Against Hydraulic Failures . . . . . . . . . . . . . . . . . . . . . . .
Supersonic liquid jets: Their generation and shock wave characteristics
As shown in Fig. 1b, the straight cone nozzle is directly connected to the exit of the pressure relief section, the liquid being re- tained in the nozzle by the use of a plastic diaphragm seal at its top and bottom.
Catalogs - Pneumatic Controls Manual
• High-temperature rolling diaphragm actuator (aluminum cover) and high-temperature plastic diaphragm retaining cup with integral air connection for 1/4 in. (6 mm) O.D. plastic tubing.
70-6926 - Commercial Components Dealer Catalog
• High-temperature rolling diaphragm actuator (aluminum cover) and high-temperature plastic diaphragm retaining cup with integral air connection for 1/4 in. (6 mm) O.D. plastic tubing.
33 3.1.1 The Plastic Diaphragm .
Since the driven section is evacuated to sub-atmospheric pressures, a light-weight plastic diaphragm can be placed on the end of the nozzle as a seal from the ambient air.