![Peptide Competition: Lysates prepared from 3T3-L1 cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either treated with lambda phosphatase (1) or left untreated (2-5), blocked with a 5% BSA-TBSTbuffer for two hours at room temperature, and incubated with ab5816 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or, the phosphopeptide immunogen (5). 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 PKA cat beta [pS338] blocks the antibody signal, thereby verifying the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_4/12373_ab5816_1.jpg)
Peptide Competition: Lysates prepared from 3T3-L1 cells were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either treated with lambda phosphatase (1) or left untreated (2-5), blocked with a 5% BSA-TBSTbuffer for two hours at room temperature, and incubated with ab5816 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), or, the phosphopeptide immunogen (5). 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 PKA cat beta [pS338] blocks the antibody signal, thereby verifying the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.