Not all ABI antibodies work in Western blot. Please see the specification sheet for each antibody to determine its suitability for Western blot use. Epitopes that depend upon tertiary protein structure are made "unrecognizable" by the SDS that is used in the separation phase of the Western blot procedure. For those antibodies that do work in Western blot, the antigen is electrophoresed on a 4 - 15% polyacrylamide minigel at a density of approximately 16.7 µg of protein per centimeter of gel width and transferred to PVDF. The membrane is blocked with 1% calfskin gelatin/5% heat inactivated Normal Goat Serum in PBS/0.05% Tween 20 and allowed to air dry while suspended. The membrane is then cut into 3 mm strips, and one strip is incubated with one milliliter of 10 µg/ml (1:100) antibody in blocking buffer for two hours at 37°C. The strip is washed in three changes of PBST20 for five minutes each and incubated with 1 milliliter of diluted Goat anti-mouse IgG-AP (BioRad 170-6520) in 100 mM Tris 150 mM NaCl pH 8.0 supplemented with 5% heat inactivated Normal Goat Serum for one hour at 37°C. The strip is washed as before and developed with BCIP/NBT (BioRad 170-6432) per manufacturer's instructions.

The dilution of 1:100 that is used in this assay is intended to check function only. It is not an "optimal" dilution that has been determined by ABI. The extent that an antibody can be diluted beyond 1:100 needs to be determined in the user's laboratory employing the user's exact methodology.