Peptide Competition: Extracts prepared from NIH3T3 cells treated with PDGF were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4^oC, then were incubated with 0.50 µg/mL ab5613 antibody for two hours at room temperature, following prior incubation with: the phosphopeptide immunogen (1), the nonphosphopeptide corresponding to the immunogen (2), a generic phosphoserine containing peptide (3), or, no peptide (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method.

Phosphatase Stripping: Extracts prepared from NIH3T3 cells not treated or treated (+PDGF) with PDGF were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either treated (lane 5) or not treated (lanes 6, 7) with lambda phosphatase, then incubated with 0.50 µg/mL ab5613 antibody for two hours at room temperature. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab5613 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: Extracts prepared from NIH3T3 cells treated with PDGF were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.50 µg/mL ab5613 antibody for two hours at room temperature, following prior incubation with: the phosphopeptide immunogen (1), the nonphosphopeptide corresponding to the immunogen (2), a generic phosphoserine containing peptide (3), or, no peptide (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. Phosphatase Stripping: Extracts prepared from NIH3T3 cells not treated or treated (+PDGF) with PDGF were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was either treated (lane 5) or not treated (lanes 6, 7) with lambda phosphatase, then incubated with 0.50 µg/mL ab5613 antibody for two hours at room temperature. After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab5613 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.