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Should protein A, G or L be used for an immunoprecipitation with this antibody (ab172)?
Asked on Nov 18 2003
I contacted the supplier of ab172, and they provided me with the following information. Chicken IgY's lack an Fc domain in the "nu" heavy chain which where proteins A and G normally bind. Consequently, protein A/G-Agarose cannot be used directly to immunoprecipitate chicken IgY's as they can mammalian IgG's. There are, however, three approaches that one can get around this limitation (the first method is detailed below): — First, one can include an extra step between chicken IgY binding to antigen and addition of protein G-Sepharose. In this step, you simply incubate the chicken IgY/antigen complex with goat anti-chicken IgY. In this way, the protein G-Agarose immunoprecipitates the goat antibody bound to the chicken antibody bound to the antigen. — Second, one can substitute "anti-chicken IgY, immobilized" (Promega Cat.#G1191, Price=$110 for 1.0 ml on pg.304 in the 1998 Promega Catalog) for protein G-Agarose. This product is a goat anti-chicken IgY antibody conjugated to agarose bead. — Third, one can conjugate your affinity-purified chicken antibodies directly to Agarose. This can be accomplished using CarboLink kit from Pierce (Cat.#44900). Solutions — Lysis buffer -- 10 mM Tris buffer (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 1.0% Triton X-100, 1.0% sodium deoxycholate, 1.0% bovine hemoglobin, 1.0 mM iodoacetamide (prepared fresh), 15 ug/ml aprotinin (prepared fresh), 1.0 mM phenylmethylsulfonyl fluoride (PMSF) (prepared fresh). — Washing buffer A -- 10 mM Tris buffer (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% Triton X-100, 0.1% bovine hemoglobin. — Washing buffer B -- 10 mM Tris buffer (pH 8.0), 150 mM NaCl, 0.02% sodium azide. — Washing buffer C -- 50 mM Tris buffer (pH 6.8) — Laemmli buffer for SDS-PAG electrophoresis Other Reagents — Unlabeled goat anti-chicken IgY — protein G-Agarose (Boehringer-Mannheim Cat.#1 719 416) Steps: 1. Lyse your 35S-labeled cells using Lysis Buffer (4C). Centrifuge the lysate at 3000 g for 15 minutes at 4C to pellet nuclei 2. Transfer the supernatant to a set of microfuge tubes and centrifuge at 10,000 g for 40 minutes to remove membranous debris. Be sure to use a refrigerated microfuge for this step, since microfuges at room temperature can get overly warm with such prolonged use. 3. Transfer the supernatant to another set of microfuge tubes and add a slurry of Protein G-Agarose to the tube. The concentration of Protein G-Agarose to be added at this step should be equivalent to that used in step # 7 below, which in turn, is based on the concentration of chicken antibodies added in step #5 below. Incubate the Protein G-Agarose slurry mixture for 2 hours at 4C with gentle agitation. 4. Centrifuge the Protein G-Agarose slurry mixture for 5 minutes at 1000 g at 4C. This removes any cellular proteins that have a natural tendency to bind Protein G-Agarose. 5. Transfer the supernantant another set of microfuge tubes. Add your affinity-purified chicken antibody to the homogenate,and incubate on ice overnight. [The amount of antibody to be added needs to be determined empirically, but is usually the same concentration as is used in enhanced chemiluminescent westerns]. 6. Add unlabeled goat anti-chicken IgY, and incubate on ice for 2 hours. [The amount of goat anti-chicken antibody should be in 2-5 fold excess over the concentration of chicken antibody, in order to assure that all of the chicken antibody-antigen complex is precipitated in subsequent steps.] 7. Add protein G-Sepharose slurry to the homogenate, and incubate 2 hours with gentle agitation in a cold room. [The amount of protein G-Sepharose added should match the molar concentration of goat anti-chicken IgG in the mixture. That is, if 1.0 ml of the protein G-Sepharose has a capacity to bind 100 femtomoles of goat antibody, and if the homogenization mixture contains a total of 200 femtomoles of goat antibody, then add 2.0 mls of the protein G-Sepharose slurry.] 8. Centifuge the slurry at 1000 g for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer A. 9. Repeat step 8. 10. Centrifuge the slurry at 1000 g for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer B. 11. Repeat step 10. 12. Centrifuge the slurry at 1000 g for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer C. 13. Remove all but a tiny volume of supernatant (<50 ul) over the agarose pellet. 14. Add Laemmli buffer. Incubate at 100C for 5 minutes. Centrifuge the slurry for 5 minutes. 15. Finally, load the supernatant onto an SDS-gel, subject the gel to electrophoresis, transfer the proteins onto a suitable membrane, and expose the membrane to an X-ray film to detect radioactive bands. Note: If you notice high backgrounds, substitute Lysis Buffer for Washing Buffer A in steps 8 and 9, above. I apologize with the delay in answering your question, and hope this information is helpful. If you have any more questions, please contact us again.
Answered on Nov 18 2003