Anti-PKC gamma (phospho T655) 抗体 (ab5796)

Peptide Competition and Phosphatase treatment: Lysates prepared from HeLa cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-11) or treated with lambda (ë) phosphatase (12), blocked with a 3% low-fat milk-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5796 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: no peptide (1, 11, 12), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-10). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase  and signals were detected using the Tropix WesternStarTM method. The data show that the peptide corresponding to PKC gamma [pT655] blocks the antibody signal while the peptides corresponding to PKC isoforms beta 1 [pT642], delta [pS645], epsilon [pT710] and eta [pT655] do not completely block the antibody signal, thereby demonstrating the specificity of the antibody. However, peptides corresponding to PKC isoforms alpha [pT638] and beta 2 [pT641] do block the antibody signal, indicating that the ab5796 antibody may cross-react with their respective sites. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.

 

Peptide Competition and Phosphatase treatment: Lysates prepared from HeLa cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-11) or treated with lambda (ë) phosphatase (12), blocked with a 3% low-fat milk-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5796 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: no peptide (1, 11, 12), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine containing peptide (3), the phosphopeptide immunogen (4), or, the phosphopeptide corresponding to the immunogen from other PKC isoforms (5-10). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStarTM method. The data show that the peptide corresponding to PKC gamma [pT655] blocks the antibody signal while the peptides corresponding to PKC isoforms beta 1 [pT642], delta [pS645], epsilon [pT710] and eta [pT655] do not completely block the antibody signal, thereby demonstrating the specificity of the antibody. However, peptides corresponding to PKC isoforms alpha [pT638] and beta 2 [pT641] do block the antibody signal, indicating that the ab5796 antibody may cross-react with their respective sites. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phosphospecific.