Immunochemistry Protocols: Western blot Antibody

Step 1

Resolve sample proteins and controls via polyacrylamide gel electrophoresis. Transfer proteins to nitrocellulose using standard methods.

Step 2

Remove the blot from the transfer apparatus and soak in TTBS (Tween-Tris Buffered Saline: 0.1% Tween-20 in 100 mM Tris-CL [pH 7.5], 0.9% NaCl) for two rinses of 15 min. each. The blot may be marked (with pencil or India Ink) for identification at this stage if desired.

Step 3

Block the blot with 10% nonfat dried milk (NFDM) freshly made in TTBS; rock on a rotating shaker for 15 min. at room temperature or overnight at 4°C.

Step 4

Rinse the blot 3 times in TTBS.

Step 5

Probe with primary antibody in TTBS/1% NFDM for 1 hr. at room temperature. Primary antibody should be diluted as specified on product data sheets. If these values are not available, use the following guidelines for initial experiments: apply antisera or ascites at 1:500 to 1:5,000, apply purified primary antibodies at a concentration of 1 ug/ml.

Step 6

Rinse the blot three times in TTBS.

Step 7

Probe the blot with an enzyme-linked secondary antibody (typically horseradish peroxidase or alkaline phosphatase) in TTBS/1% NFDM for 30 min. at room temperature. Review instructions included with the secondary antibody to determine the appropriate dilution to use.

Step 8

Rinse excess secondary antibody from the blot with 3 rinses in 20-50 ml TTBS for 5 min. each. The blot is now ready for use with standard colorimetric or chemiluminescent detection reagents.

Considerations: