Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated IRS 1. The final product is generated by affinity chromatography using an IRS 1-derived peptide phosphorylated at serine 312.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
WB: We recommend a starting dilution of 1/1000. Predicted molecular weight: 165 kDa.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
May mediate the control of various cellular processes by insulin. When phosphorylated by the insulin receptor binds specifically to various cellular proteins containing SH2 domains such as phosphatidylinositol 3-kinase p85 subunit or GRB2. Activates phosphatidylinositol 3-kinase when bound to the regulatory p85 subunit.
Polymorphisms in IRS1 may be involved in the etiology of non-insulin-dependent diabetes mellitus (NIDDM) [MIM:125853].
Serine phosphorylation of IRS1 is a mechanism for insulin resistance. Ser-312 phosphorylation inhibits insulin action through disruption of IRS1 interaction with the insulin receptor. Phosphorylation of Tyr-896 is required for GRB2-binding.
Western blot - IRS1 (phospho S312) antibody (ab4865)
Predicted band size : 165 kDa
Peptide Competition and Phosphatase Stripping: Extracts prepared from CHO-T cells transiently transfected with wild-type human IRS 1 and treated with TPA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were treated with lambda phosphatase (1) or left untreated (2-6), blocked with a 5% BSA-TBST buffer overnight at 4oC, then incubated with 0.50 µg/mL ab4865 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1-3), the non-phosphopeptide corresponding to the immunogen (4), a generic phosphoserine containing peptide (5), or, the phosphopeptide immunogen (6). 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 ab4865 blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.
Dávila D & Torres-Aleman I Neuronal Death by Oxidative Stress Involves Activation of FOXO3 through a Two-Arm Pathway that Activates Stress Kinases and Attenuates Insulin-like Growth Factor I Signalling. Mol Biol Cell : (2008).
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