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Western Blotting using HRP-Labeled Secondary

Solutions

  • PBS – sodium phosphate-buffered (100 mM; pH 7.2) isotonic (0.9% NaCl, w/v) saline
  • Antibody dilution buffer (PBS with 0.1% non-ionic detergent, such as Triton X-100 or Tween-20)

Other Reagents

  • Horseradish Peroxidase-labeled goat anti-chicken IgY (Aves Labs, Catalog # H-1004)
  • BlokHen® (Aves Catalog # BH-1001)
  • SuperSignal® Substrate for Western Blotting (Pierce Chemical Company, Catalog #34080)

Steps

  1. Run your SDS-polyacrylamide Laemelli gel and blot the protein bands onto nitrocellulose membrane, following standard methods (see chapter 12 of Harlow and Lane, 1988). Be sure NOT to use PVDF or other nylon-based membranes, as chicken antibodies tend to bind non-specifically, contributing to higher backgrounds.
  2. Block non-specific sites on your membrane by incubating the membrane for 30 min at room temperature with BlokHen® (diluted 1:10 with water) using gentle agitation.
  3. Wash the blot three times in PBS with 0.05% Tween-20 at room temperature. Each wash should be at least 10 minutes with gentle agitation.
  4. Incubate your blot for at least 1 hour at room temperature with your chicken antibody diluted in “antibody dilution buffer.” Optimal dilutions of chicken antibody may be as low as 1:500 or as high as 1:50,000 depending on the immunogenicity of the antigen. Generally, the longer the period of incubation, the better (provided that the blot doesn’t dry out) — even overnight incubations are fine.
  5. Wash the blot in PBS, as described above (step 3).
  6. Incubate your blot for 1 hour at room temperature with a 1:5,000 to 1:10,000 dilution (in "antibody dilution buffer") of horseradish peroxidase-labeled goat-anti-chicken IgY with gentle agitation.
  7. Wash the blot in PBS, as described above (step 3).
  8. Develop the blot for 5-10 minutes at room temperature using SuperSignal® HRP Substrate Working Solution.
  9. Place the blot against X-ray film and exposure from 10 seconds to 5 minutes (depending on the intensity of the signal).
  10. Develop the X-ray film as you normally would.

Notes

  • Chicken antibodies tend to display higher backgrounds on polyvinylidene fluoride (PVDF) and other nylon-based membranes. For this reason, we recommend using nitrocellulose membranes for western blotting applications.

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.