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Western blot - Anti-IKB alpha (phospho S32 + S36) antibody (ab5682)
Peptide Competition and Induction: Extracts prepared from ALLN pretreated Jurkat cells left unstimulated (1, 2) or stimulated with TNF alpha (3 7) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer for one hour at room temperature and incubated with anti-IKBa pan (1, 3) or ab5682 antibody (2, 4-7) overnight at 4°C in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-4), the non phosphopeptide corresponding to the immunogen (5), a generic phosphoserine-containing peptide (6), or, the phosphopeptide immunogen (7). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that the phosphorylation of IKBa is induced by the addition of TNF alpha and that the peptide corresponding to IKBa [pSpS32/36] blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that treatment with TNF alpha leads to degradation of IKBa protein (compare lanes 1 and 3), as expected, even in the presence of ALLN.
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