Anti-PKC eta (phospho T655) 抗体 (ab5798)

Peptide Competition and Phosphatase Treatment Lysates prepared from Jurkat cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-9) or treated with lambda  phosphatase (10), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5798 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 10), 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 (5-9). After washing, membranes were incubated with goat  (ab’)2 anti-rabbit IgG HRP-conjugate  and bands were detected using the Pierce SuperSignalTM method. The data show that the peptide corresponding to PKC eta  [pT655] blocks the antibody signal. The antibody signal was not blocked by the peptides corresponding to PKC isoforms alpha [pT638], beta 1 [pT642], beta 1 [pT641], gamma [pT655] and zeta [pT560], thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific. 

Peptide Competition and Phosphatase Treatment Lysates prepared from Jurkat cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-9) or treated with lambda phosphatase (10), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5798 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 10), 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 (5-9). After washing, membranes were incubated with goat (ab’)2 anti-rabbit IgG HRP-conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that the peptide corresponding to PKC eta [pT655] blocks the antibody signal. The antibody signal was not blocked by the peptides corresponding to PKC isoforms alpha [pT638], beta 1 [pT642], beta 1 [pT641], gamma [pT655] and zeta [pT560], thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.