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Western Blotting Using Alkaline Phosphatase-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

  • Aves Labs Alkaline Phosphatase-labeled Goat Anti-chicken IgY (Catalog #AP-1001)
  • Western Blue® Stabilized AP Substrate (Promega, Catalog #S3841 (this is a stabilized mixture of BCIP (5-bromo-4-chloro-3-indolyl-1-phosphate) and NBT (nitro blue tetrazolium)))
  • Aves Labs BlokHen® (Catalog #BH-1001)

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 minutes 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 one (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 the result, provided that the blot doesn’t dry out. Even overnight incubations are fine.
  5. Wash the blot in PBS, as described in Step 3 above.
  6. Incubate your blot for one (1) hour at room temperature with a 1:3000 dilution in “antibody dilution buffer” of Alkaline Phosphatase-labeled Goat Anti-chicken IgY (Aves Labs Catalog #AP-1001) with gentle agitation.
  7. Wash the blot in PBS, as described in Step 3 above.
  8. Develop the blot for 5-20 minutes at room temperature using Western Blue Stabilized AP Substrate®.

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.