Western Blot Protocol (NSB752)

Protocol used for the datasheet image:

Peptide Competition Human recombinant Tau untreated (1) or treated with GSK-3b (1 ug per ug Tau) for 45 minutes (2-5) was added to background extracts, 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, and then incubated with the Tau [pS396] antibody in a 1% BSA-TBST buffer for two hours at room temperature, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab)2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignal(TM) method. The data show that only the peptide corresponding to Tau [pS396] blocks the antibody signal, demonstrating the specificity of the antibody.