Affinity-purified rabbit anti

Affinity-purified rabbit anti-RIP antibody solution 6 mg in 6 mL of loading buffer. 

Prepare a solution containing affinity purified rabbit anti-RIP antibody at a concentration of approx 1 mg/mL of loading buffer and pass through a 0.22-pm filtration unit... 

Substrate buffer 12 mL of O.lMcitric acid (stock solution stored at 4°G), 13 mL of 0.2MNa2HPO4 (stock solution) and 25 mL of distilled water. Check that the pH of the solution is 5.0. Add 20 mg of OPD and shake to dissolve. Then add 10 pL of 100 vol H2O2 solution and mix well. Prepare this solution shortly before use and cover to protect from light Affinity-purified rabbit anti-RIP antibody solution in carbonate/bicarbonate buffer, 20-40 pg per microtiter plate. 

Incubate in affinity-purified rabbit anti-cannabinoid receptor IgG overnight at 4°C (in a humidified chamber). A first immunolabeling should include a titration series of the specific affinity-purified antibody at dilutions of 1 5-1 20 to establish the optimal dilution. This titration series is required to optimize the signal-to-noise ratio of immunolabeling. Concnrrent incubations should be carried out in non-immune rabbit IgG (at similar dilutions) from the primary antibody donor species to serve as negative controls. 

Alternative Approach Using Affinity-Purified Rabbit Anti -H2AX... 

Figure 5. Detection and quantitation of y-H2AX foci at low radiation doses. Human fibroblasts were immunostained with affinity-purified rabbit anti-y-H2AX antibody and counterstained for DNA with DAPI. Gamma irradiation...

Anti-cannabinoid CB2 Affinity-purified rabbit IgG directed against amino acids 20-33 at the amine terminus of CB2 reacts with human Calbiochem-Novabiochem Corp., La Jolla, CA (cat no. 216407)... 

Quantitative studies of antiidiotypic antibodies directed to specifically purified rabbit anti-p-azobenzoate antibody indicated that the combining site of the antibenzoate antibody is part of, or close to a major idiotypic determinant (84). The reaction of I-labeled F(ab )2 fragments of antibenzoate antibody with each of six antiidiotypic antisera tested was significantly inhibited by a large number of homologous haptens, i.e., by benzoate derivatives. There was a close correlation between the affinity of a hapten for the antibody and its capacity to prevent the combination of antibenzoate antibody with antiidiotypic antibody. Compounds unrelated to benzoate were not inhibitory. As much as 69% inhibition was observed when the hapten of highest affinity, p-(p -hydroxy)phenylazobenzoate, was tested at a concentration of 1.6 X 10- M. 

Cholera toxin B subunit-biotin labeled (lyophilized powder, biotin content 0.9mol/mol protein), peroxidase-labeled IgG anti-rabbit antibody (HRP-Ab, from goat, protein content 0.8mg/ml, affinity isolated antibody), anti-cholera toxin (from rabbit, protein content 48mg/ml, purified toxin from Vibrio cholerae), biotin monoclonal anti-rabbit IgG -y-chain specific (from mouse, protein content 4.2mg/ml), glucose oxidase-biotinamidocaproyl labeled (GOX-B, from Aspergillus niger, lyophilized powder containing 40-70% protein, 137 U/mg), polyoxyeth-ylene-sorbitan monolaurate (Tween 20), bovine serum albumin (fraction... 

Both E. coli and T4 thioredoxin have recently been purified by immu-noabsorbent affinity chromatography. The specific immunoadsorbents were prepared by coupling the a-globular fractions of rabbit anti-thio-redoxin antisera to Sepharose. This technique makes it possible to isolate both thioredoxins from phage-infected cells by two consecutive chromatography steps on the specific adsorbents (125, 126). 

Cover tissue slice with the primary anti-cannabinoid receptor antibody (see Table 1). It is preferable initially to use at least four dilutions of affinity purified antibody (1 10, 1 25, 1 50, 1 100) in blocking buffer. Use a 1 25 dilution of normal rabbit... 

Cover tissue slice with the primary rabbit IgG anti-cannabinoid receptor antibody. It is preferable initially to use at least four dilutions of each affinity-purified antibody (1 10, 1 25, 1 50, 1 100) in blocking buffer. Use a 1 25 dilution of normal rabbit IgG (1 mg/mL) in blocking solution as a negative control. Incubate for 1 h at room temperature in a hunudified chamber. Do not let the sections dry out in this or subsequent steps. Ideally, a negative control should consist of preimmune IgG derivative from the same rabbit which was used to produce the anti-cannabinoid receptor antibody. However, when using commercially available anti-cannabinoid receptor antibodies, such preimmune IgG may not be available and normal rabbit IgG can be used as a control. 

Incubate sections overnight in a refrigerator (4°C) on a shaker with affinity-purified primary rabbit anti-cannabinoid receptor antibody (see Table 1). Use the affinity-purified antibody at a 1 1000-1 5000 dilution of a stock antibody preparation (1 mg/mL) in a final volume of 5 mL PBS supplemented with 1% NGS. Note For immunoelectron miCToscopy it is recommended that affinity-purified antibody be used (see Note 1). 

The specific activity of affinity-purified anti-poly(ADP-ribose) antibody was ten times greater than that of the 7-globulin fraction the specific activity was defined as the amount of protein required to give 1 A -os unit (antibody titer) under certain conditions. The poly(ADP-ribose) binding specificity of the rabbit antibody [8] was confirmed by both direct and competitive ELISA (data not shown). 

Anti-a-D-mannose antibodies have been isolated from the serum of rabbits immunized with the glycoconjugate of a-D-mannose and bovine serum albumin [85], The antibodies were purified by affinity chromatography with adsorption on a mannosyl-Sepharose column and elution with a-D-mannose or methyl a-D-mannoside. Such antibodies should be especially useful for studying the detection of diseases due to the appearance of abnormal glycoproteins that contain mannose polymers. 

Antibodies with specificity for specific carbohydrate residues of antigens have been isolated from serum of rabbits immunized with carbohydrate containing antigens. Eighteen such antibodies were purified by affinity chromatography on adsorbents with ligands of carbohydrate residues. Electrofocusing results showed that all the antibodies occurred in multiprotein forms. A number of anti-carbohydrate antibodies have had useful applications. In the future additional advances with anti-carbohydrate antibodies should be made and these can lead to developments of new and improved products and processes. Some of the applications are summarized ... 

Purification of Immunoglobulins and Anti-immunoglobulins. The IgG fraction of an antiserum can be purified according to any of a number of established techniques. If the antiserum was raised in rabbits, the most convenient purification procedure to use is affinity chromatography on protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). Excellent yield and purity is obtained by following the recommendations of the manufacturer. 

---