Peptide Competition and Phosphatase Stripping:

Extracts of NIH3T3 cells stably expressing c-Src Y527F mutant protein were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was left untreated (1-4) or treated with lambda (λ) phosphatase (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the cortactin [pY466] (mouse) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphotyrosine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase (cat.# ALI4405) and signals were detected using the Tropix WesternStar™ method.

Extracts of NIH3T3 cells stably expressing c-Src Y527F mutant protein were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was left untreated (1-4) or treated with lambda (λ) phosphatase (5), blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the cortactin [pY466] (mouse) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphotyrosine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG alkaline phosphatase (cat.# ALI4405) and signals were detected using the Tropix WesternStar™ method.

Related Products

Related Images