Peptide Competition
Extracts prepared from Jurkat E6.1 cells were left unstimulated (1) or stimulated (2-5), resolved by SDS-PAGE on a 10% polyacrylamide gel, and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C, then were incubated with 0.35 µg/ml phospho LAT (Tyr 132) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the nonphosphopeptide corresponding to the immunogen (3), a generic phosphotyrosine 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 phospho LAT (Tyr 132) completely blocks the antibody signal, thereby demonstrating the specificity of the antibody, and stimulation-induced tyrosine phosphorylation of phospho LAT (Tyr 132).