Anti-IRS1 (phospho S312) antibody (ab4865)

Peptide Competition and Phosphatase Stripping: Extracts prepared from CHO-T cells transiently transfected with wild-type human IRS 1 and treated with TPA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were treated with lambda phosphatase (1) or left untreated (2-6), blocked with a 5% BSA-TBST buffer overnight at 4^oC, then incubated with 0.50 µg/mL ab4865 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-3), the non-phosphopeptide corresponding to the immunogen (4), a generic phosphoserine containing peptide (5), or, the phosphopeptide immunogen (6). 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 ab4865 blocks the antibody signal, thereby demonstrating the specificity of the anti