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Up-regulation and Antibody-Peptide Competition:
Peptide Competition and Phosphatase Stripping: Extracts of CHO-K1 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, either left untreated or treated with lambda phosphatase, then incubated with 0.75 µg/ml GRK2 [pS670] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide, the non-phosphopeptide corresponding to the phosphopeptide immunogen, a generic phosphoserine-containing peptide, or the phosphopeptide immunogen. After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG alkaline phosphatase (Cat. no. ALI4405) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to GRK2 [pS670] blocks the antibody signal, 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 Stripping: Extracts of CHO-K1 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, either left untreated or treated with lambda phosphatase, then incubated with 0.75 µg/ml GRK2 [pS670] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide, the non-phosphopeptide corresponding to the phosphopeptide immunogen, a generic phosphoserine-containing peptide, or the phosphopeptide immunogen. After washing, the membrane was incubated with goat F (ab')2 anti-rabbit IgG alkaline phosphatase (Cat. no. ALI4405) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to GRK2 [pS670] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.](http://tools.thermofisher.com/content/sfs/gallery/low/44202a-Final.jpg)
