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Does R & D Antibodies have a Quality Policy?Yes! QUALITY POLICY How do I store my antibodies?Monoclonal antibodies and polyclonal antisera must be handled and stored properly to insure that these proteins are not denatured. Adverse storage conditions can destroy the binding capacity of the antibodies. RIA grade antibodies purchased in bulk should be diluted to 1000 RIA tubes/ml in 1.0% BSA in PBS, aliquoted into single use portions and either stored frozen or lyophilized and stored at 4° C. Do not subject antibodies to repeated freeze/thaw cycles. Dilute or rehydrate the antibodies to 10 RIA tubes/ml in RIA buffer prior to use. After dilution of bulk RIA grade or rehydration of pretitered RIA grade antibodies to 10 RIA tubes/ml in RIA buffer, the solution should be stored at 4° C. Do not freeze solutions containing antibodies and detergents, such as Triton X-100 or Tween: these solutions should be refrigerated for short term shorage. Histochemical grade antibodies should be diluted with PBS that contains either 1.0% BSA or 1% normal goat serum, aliquoted into single use portions, and stored frozen at -20° C: do not repeatedly freeze and thaw. For short term storage, antibody containing solutions can be refrigerated. The addition of 0.01% sodium azide or 0.01% thimerosal to inhibit microbial growth can be used to prolong the length of time antibody containing solutions can be refrigerated: these chemicals should only be used if they will not effect the utilization of the antibodies.
How does R & D Antibodies perform western blots using mouse monoclonal antibodies?1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels, such as 7.5% or 10% gels) and electrophoretic transfer to PVDF membrane, the membrane is blocked overnight with 4% normal goat serum using TBS/Tween-20 buffer as diluent. 2. Wash x 2 for 5 min with TBS/Tween-20. 3. Apply the mouse monoclonal antibody after dilution to an appropriate volume (at least 1:20 for culture supernatant or 1:500 for ascites - see Note below). Use 2% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. Let the primary antibody bind for 2-4 hours. 4. For blocked antibody controls, add 150 nmoles of the peptide immunogen or 150 μgm of the protein antigen to 6.0 ml of the diluted monoclonal antibody. Preincubate for 60 min. before applying to the membrane. This should be sufficient for 3 blocked control lanes. DO NOT ADD THE PEPTIDE OR PROTEIN TO THE STOCK MONOCLONAL ANTIBODY. 5. Wash x 3 for 5 min with TBS/Tween-20. 6. Apply affinity purified HRP-goat anti-mouse IgG or IgM antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20 as buffer. Incubate 1-2 hours. When we need increased sensitivity, we use biotinylated horse anti-mouse IgG or IgM (at 1:2000 dilution) in conjunction with ABC complex (at 1:1000) instead of the HRP-conjugated second antibody and substitute normal horse serum for the normal goat serum in all steps of the procedure. 7. Wash x 4 for 5 min in TBS/Tween-20. 8. Develop color using the enhanced DAB reaction. Note : It is important to conduct initial titration experiments with the monoclonal antibody on the sample in the western immunoblot format. One wants a good signal (i.e. band on the membrane), but it is important for blocking experiments not to have an excess of antibody because it will be impossible to block all of the antibody binding to the membrane by preincubating with the peptide or protein. If one blocks 95% of the binding with the antigen, then 5% of the antibody is still available to bind. In antibody excess this will be enough to produce a band on the membrane.
How does R & D Antibodies perform western blots using rabbit polyclonal anti-peptide antibody?1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels, such as 7.5% or 10% gels) and electrophoretic transfer to PVDF membrane, the membrane is blocked overnight with 4% normal goat serum using TBS/Tween-20 buffer as diluent. 2. Wash x 2 for 5 min with TBS/Tween-20. 3. Apply the rabbit polyclonal antibody after dilution to at least 1:200 (higher dilutions may be needed - see Note below). Use 2% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. Let the primary antibody bind for 2-4 hours. 4. For blocked antibody controls, add 150 nmoles of the peptide immunogen or 150 μgm of the protein antigen to 6.0 ml of the diluted rabbit polyclonal antibody. Preincubate for 60 min before applying to the membrane. This should be sufficient for 3 blocked control lanes. DO NOT ADD THE PEPTIDE OR PROTEIN TO THE STOCK POLYCLONAL ANTIBODY. 5. Wash x 3 for 5 min with TBS/Tween-20. 6. Apply affinity purified HRP-goat anti-rabbit IgG antiserum diluted 1:2500 (dilution may vary depending upon supplier) in 2% normal goat serum in TBS/Tween-20. Incubate 1-2 hours. When we need increased sensitivity, we use biotinylated horse anti-rabbit IgG (at 1:2000) in conjunction with ABC complex (at 1:1000) instead of the HRP-conjugated second antibody and substitute normal horse serum for the normal goat serum in all steps of the procedure. 7. Wash x 4 for 5 min in TBS/Tween-20. 8. Develop color using the enhanced DAB reaction. Note : It is important to conduct initial titration experiments with the antibody on the sample in the western immunoblot format. One wants a good signal (i.e. band on the membrane), but it is important for blocking experiments not to have an excess of antibody because it will be impossible to block all of the antibody binding to the membrane by preincubating with the peptide or protein. If one blocks 95% of the binding with the antigen, then 5% of the antibody is still available to bind. In antibody excess this will be enough to produce a band on the membrane.
How does R & D Antibodies perform ICC experiments on cells and tissues?Initially, one should perform a titration experiment to optimize the signal to noise ratio especially if peptide or protein blocking experiments are to be conducted. However, we have successfully used our monoclonal antibodies as culture supernatants, our monoclonal antibodies as ascites fluids, and our polyclonal antibodies at dilutions of at least1:50, 1:500 and 1:1000, respectively (see specific data sheet for individual antibody). Dilute the stock antibody in PBS, pH 7.2, containing either 0.1% BSA or 1% normal goat serum. For cells cultured on slides or coverslips We wash the cells 4 times in PBS pH 7.2, rinse once quickly in distilled water, and drain well. We fix the cells with either 70% ice cold acetone or neutral buffered formalin for 10 min, air dry and store them frozen. We always allow the slides to warm to room temperature before using. If the cells were fixed with organic solvents, such as 70% acetone, 95% ethanol, or methanol, then they do not need permeabilizing. However, if fixed with formalin, formaldehyde, or glutaraldehyde, then the cells must be permeabilized before being exposed to the primary antibody. To permeabilize the cells, soak the fixed cells for 20 mins in PBS that contains 0.1% Triton X-100. Before applying the primary antibody, block non-specific binding by soaking the cells in 1% normal goat serum in PBS for 20 min. For blocked controls preincubate the diluted antibody with either 50 nmole of peptide or 50 μgm of protein per ml of diluted antibody solution for 60 minutes. Apply either the diluted primary antibody or the peptide/protein blocked antibody for 1 - 2 hours, wash 4 times in PBS, and then apply the diluted enzyme or fluorochrome labeled goat anti-mouse IgG, -mouse IgM, or -rabbit IgG second antibody for 60 min. Wash 4 times in PBS and rinse once quickly with distilled water. Mount a cover slip using a water and glycerol based mounting medium. For tissue sections We routinely test two different fixatives: 3.0% freshly depolymerized paraformaldehyde and PLP* (see below). All incubations and washing steps should be done with the tissue in constant gentle motion on an orbital shaker. We use 60 μm sections of either 3.0% paraformaldehyde or PLP* fixed whole rat brain which are cut on a freezing microtome, and washed x 4 for 15 min with PBS to remove residual fixative. Sections are then blocked and premeabilized at room temperature for 30 minutes in 1.5% normal goat serum in PBS containing 0.2% Triton X-100. Incubation with the primary antibody is performed at 4° C for 48 hours. All remaining steps are performed at room temperature. Following the incubation with the primary antibody, the sections are washed for 10 minutes in PBS to remove residual antibody before they are incubated with biotinylated goat anti-mouse IgG, -mouse IgM, or -rabbit IgG for 1 hour. After a 10 min wash in PBS, the sections are incubated with ABC reagent for 1 hour which is followed by a final wash in PBS. The sections are then transferred to 0.05 M Tris buffer, pH 7.6, and incubated for 3 minutes in 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H2O2 to visualize the reaction product. The reaction is stopped in Tris buffer and the sections transferred first to PBS then to 10 mM sodium acetate before being mounted, cleared, and coverslipped. Mounted sections are visually examined with a microscope. Control sections are incubated either with pre-immune rabbit serum or with antiserum blocked by prior incubation with the immunogen. * PLP - Periodate/Lysine/Paraformaldehyde - Ref: McLean and Nakane (1974) J Histochem Cytochem , 22 , 1077-1083 2% Paraformaldehyde, 0.1 M L-Lysine, 0.1 M Sodium meta-periodate in 0.1 M Na 2HPO4 (pH 7.4) Note : It is important to conduct initial titration experiments with the antibody on the sample. One wants good immunostaining, but it is important for blocking experiments not to have an excess of antibody because it will be impossible to block all of the antibody binding by preincubating with the peptide or protein. If one blocks 95% of the binding with the antigen, then 5% of the antibody is still available to bind. In antibody excess this will be enough to produce immunostaining of the cells or tissue.
What scientific meetings/exhibits will R & D Antibodies be exhibiting at in the next year?No exhibits are planned at this time. How can I contact R & D Antibodies?Customer and Technical Service hours are 8:30 a.m. to 5:30 p.m. Monday through Friday Pacific Coast Time. You may contact us by telephone, facsimile, e-mail or regular mail as detailed below. We strive to respond to all correspondence within one business day. Please feel free to contact us regarding new products that you are interested in obtaining from a commercial source. We always appreciate hearing from our customers regarding their specific uses of our products so don't hesitate to contact us.
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