Simple Western Blot Protocol
Perform SDS-PAGE and transfer to nitrocellulose membrane according to manufacturers directions.
1. Block membrane 10 minutes with Pierce Superblock or similar reagent
2. Incubate 30 minutes with primary antibody at 2 ug/ml in TBS-BSA
3. Wash 10 minutes with TBST
4. Incubate 30 minutes with secondary antibody at 1:3000 or at manufacturers recommended dilution in TBS-BSA
5. Wash 10 minutes with TBST
6. Wash 10 minutes with TBS
7. Incubate with appropriate substrate until bands develop
8. Rinse with di H20 and dry
Optimal working dilutions depend on assay conditions and should be determined by the individual laboratory.
TBS – 0.1 M Tris; 0.15 M NaCl; pH 7.4
TBS-BSA – TBS with 3% Bovine Serum Albumin
TBST – TBS with 0.5% Tween
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