Western blot using Rabbit anti Cofilin [pS3] polyclonal antibody:
Peptide Competition and Phosphatase Treatment: Lysates prepared from MDCK cells treated with staurosporine (1) or left untreated (2-6) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-5) or treated with lambda (λ) phosphatase (6), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with the cofilin [pS3] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4) or, the phosphopeptide immunogen (5). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate (Cat. # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that only the peptide corresponding to cofilin [pS3] blocks the antibody signal, and that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.