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Immunoprecipitation

Chicken IgY’s lack the Fc domain where proteins A and G normally bind. Consequently, protein A/G-Agarose or -Sepharose cannot be used to immunoprecipitate chicken IgY’s. There are, however, three alternative approaches to get around this limitation:

  1. Substitute PrecipHen for protein G-Agarose. This product is a goat anti-chicken IgY antibody conjugated to agarose beads.
  2. 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.
  3. Conjugate your affinity-purified chicken antibodies directly to Agarose. This can be accomplished using CarboLink kit from Pierce (Catalog #44900).

Reagents

  • 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
  • PrecipHen® (Agarose-coupled goat anti-chicken IgY, Aves Labs, Catalog #P-1010)

Steps

  1. Lyse your cells or tissue using Lysis Buffer (4C). Centrifuge the lysate at 3000 g for 15 minutes at 4C to pellet nuclei and other cellular debris.
  2. Transfer the supernatant to a set of microfuge tubes and centrifuge at 10,000 g for 40 minutes at 4C to remove smaller membranous material.
  3. Transfer the supernatant to another set of microfuge tubes and add a slurry of PrecipHen® to the tube. Typically, a volume of 200 ul (a packed bed volume of 100 ul plus 100 ul of PBS) is sufficient for this step. Incubate this mixture for 30 minutes at 4C with gentle agitation to keep the PrecipHen® in suspension. Finally, centrifuge the PrecipHen® to remove any proteins in your sample that happen to bind non-specifically to either the agarose or the goat antibody.
  4. Transfer the supernantant to another set of microfuge tubes, and add your primary chicken antibody to the tube. Incubate 1-2 hours on ice. [NOTE: In most cases, the primary antibody needs to be affinity-purified. This is because only a small fraction (less than 1%) of the antibody in purified IgY fractions is likely to be against the antigen of interest. Consequently, only a small fraction of the antibodies in IgY fractions contribute to specific binding of the protein of interest, whereas the other antibodies contribute to non-specific binding. In contrast, 100% of affinity-purified antibodies recognize the protein of interest, greatly improving signal-to-noise ratios.]
  5. Add a slurry of PrecipHen® to the tube. [NOTE: The amount of PrecipHen to be added depends on the total amount of chicken IgY present in the tube. Since the binding capacity of 1.0 mL PrecipHen (packed volume) is 1 mg of chicken IgY, one should add approximately twice the binding capacity of the PrecipHen. In other words, if you added 50 micrograms of primary chicken IgY to each microfuge tube in step 4 above, you would want to add 100 microliters (packed volume) of Preciphen to that tube. Since PrecipHen is a 1:1 slurry in PBS, this means that you would add a 200 ul slurry volume to each tube.]
  6. Incubate for 3 hours – overnight at 4C with gentle agitation.
  7. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer A. Incubate for 5 minutes with gentle agitation.
  8. Repeat step 7.
  9. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer B. Incubate for 5 minutes with gentle agitation.
  10. Repeat step 9.
  11. Centifuge the slurry in a refrigerated microfuge for 5 minutes at 4C. Discard the supernatant. Resuspend the slurry in Washing Buffer C. Incubate for 5 minutes with gentle agitation.
  12. Repeat step 11.
  13. Centrifuge the slurry in a refrigerated microfuge at 4C. Discard the supernatant.
  14. Add Laemmli buffer. Place the tube in boiling water for 5 minutes. Be sure to poke a small hole in the cap of the tube to prevent built-up of gases.
  15. Vortex the microfuge tube briefly, and then centrifuge it again for 5 minutes at 4C in a microfuge.
  16. Finally, load the supernatant onto an SDS-polyacrylamide gel, subject the gel to electrophoresis, and transfer the proteins onto a suitable membrane. This membrane is now ready for western blotting with a second antibody against the protein of interest.

Notes

  • If you notice high backgrounds, substitute Lysis Buffer for Washing Buffer A in steps 7 and 9, above.

Further Reading on Methods

These methods have been adapted from a variety of sources, including:

  • “Antibodies: A Laboratory Manual” by Harlow and Lane (Cold Spring Harbor Press, 1988);
  • “Current Protocols in Molecular Biology” by Ausubel et al. (Wiley & Sons, 1997); and
  • various Specification Sheets offered by manufacturers.

We highly recommend the manual by Harlow and Lane, which provides general information on antibodies.