HeLa cell lysates (A) and HeLa cell lysates treated with alkaline phosphatase (B) were resolved by SDS-PAGE on a 4-20% Tris-glycine gel. The proteins were then transferred to PVDF membrane. Membranes were incubated with 1 µg/mL ab4774. After washing, membranes were incubated with goat  (ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method. HeLa cell lysates (A) and HeLa cell lysates treated with alkaline phosphatase (B) were resolved by SDS-PAGE on a 4-20% Tris-glycine gel. The proteins were then transferred to PVDF membrane. Membranes were incubated with 1 µg/mL ab4774. After washing, membranes were incubated with goat (ab’)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar detection method.

Extracts of HeLa cells were resolved by SDS-PAGE on a 4-20% 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 (1-4) or treated with lambda (?) phosphatase (5), and then incubated with the eIF4E (phospho S209) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1,5), the nonphosphopeptide corresponding to the phosphopeptide immunogen (2), a generic phosphoserine-containing peptide (3), or the phosphopeptide immunogen (4). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG alkaline phosphatase and signals were detected using the Pierce SuperSignal™ method.

The data show that only the peptide corresponding to eIF4E (phospho S209) blocks the antibody signal, demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody i