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    Why Use Chicken IgY?

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    1. Higher titres against highly conserved mammalian gene products

    2. Double immunostaining is easier to perform

    3. Animal-friendly -- we purify the antibodies from eggs, not serum

    4. Large quantities of antibody - faster and cheaper

    5. Nearly unlimited quantities (again, because the antibodies come from eggs)

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Western Blotting Using Alkaline Phosphatase-Labeled Secondary (Cat.# AP-1001)

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

Alkaline Phosphatase-labeled goat anti-chicken IgY (Aves Labs, 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)]

BlokHen® (Aves Labs, Catalog # BH-1001 — for more information about BlokHen click here.)

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 1:3000 dilution (in “antibody dilution buffer”) of Alkaline Phosphatase-labeled goat-anti-chicken IgY (Aves Cat. #AP-1001 with gentle agitation.

7. Wash the blot in PBS, as described above (step 3).

8. Develop the blot for 5-20 minutes at room temperature using Western Blue Stabilized AP Substrate®.

* These methods have been adapted from a number 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. Other useful resources on antibodies can be found on our Links to Other Websites page.