Peptide Competition and Phosphatase Stripping Total cell lysates (1) or immunoprecipitates (2-7) prepared from Hek293 cells overexpressing c-Raf and stimulated with EGF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either untreated (1-6), or treated with lambda (λ) phosphatase (7), blocked with a 5% BSA-TBST buffer overnight at 4oC, then incubated with 0.50 µg/mL c-Raf [pS259] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2, 6, 7), 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 alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to c-Raf [pS259] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.