![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 (1-13) or treated with lambda (ë) phosphatase (14), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5797 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: the phosphopeptide corresponding to the immunogen from other PKC isoforms (1-9), the phosphopeptide immunogen (10), the non-phosphopeptide corresponding to the immunogen (11), a generic phosphothreonine containing peptide (12) or, no peptide (13, 14). After washing, membranes were incubated with goat F(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 gamma [pT674] blocks the antibody signal. The peptides corresponding to PKC alpha [pT65](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/12640_ab5797_1.jpg)
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 (1-13) or treated with lambda (ë) phosphatase (14), blocked with a 5% BSA-TBST buffer overnight at 4°C, and incubated with 0.50 µg/mL ab5797 antibody for two hours at room temperature in a 3% BSA TBST buffer, following prior incubation with: the phosphopeptide corresponding to the immunogen from other PKC isoforms (1-9), the phosphopeptide immunogen (10), the non-phosphopeptide corresponding to the immunogen (11), a generic phosphothreonine containing peptide (12) or, no peptide (13, 14). After washing, membranes were incubated with goat F(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 gamma [pT674] blocks the antibody signal. The peptides corresponding to PKC alpha [pT65
WB from review by Todd O'Buckley submitted 9 April 2004.