Western blot analysis of extracts from CHO IR/IRS-1 cells ( transfected with insulin receptor and IRS-1), untreated or insulin-treated (100 nM for 5 min), showing an increase in phospho-IRS-1 (Ser302) with insulin stimulation, using Phospho-IRS1 (Ser302) Antibody. To confirm phospho-specificity of the antibody, a duplicate membrane was treated with calf intestinal alkaline phosphatase (CIP) after Western transfer.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
Phospho-IRS-1 (Ser 302) Antibody detects endogenous levels of IRS-1 only when phosphorylated at Ser302 of mouse IRS-1 or Ser307 of human IRS-1. This antibody does not detect IRS-1 phosphorylated at other sites.
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser 302 of mouse IRS-1. Antibodies are purified by protein A and peptide affinity chromatography.
Insulin receptor substrate 1 (IRS-1) is one of the major substrates of the insulin receptor kinase (1). IRS-1 contains multiple tyrosine phosphorylation motifs that serve as docking sites for SH2-domain containing proteins that mediate the metabolic and growth-promoting functions of insulin (2-4). IRS-1 also contains over 30 potential serine/threonine phosphorylation sites. Ser307 of IRS-1 is phosphorylated by JNK (5) and IKK (6) while Ser789 is phosphorylated by SIK-2, a member of the AMPK family (7). The PKC and mTOR pathways mediate phosphorylation of IRS-1 at Ser612 and Ser636/639, respectively (8,9). Phosphorylation of IRS-1 at Ser1101 is mediated by PKCθ and results in an inhibition of insulin signaling in the cell, suggesting a potential mechanism for insulin resistance in some models of obesity (10).
Ser302 in rat/mouse IRS-1 (corresponding to Ser307 of human IRS-1) is phosphorylated rapidly during insulin stimulation and has a postive role in IRS-1 tyrosine phosphorylation. Inhibition of Ser302 phosphorylation by short-term amino acid/glucose starvation correlates with a decrease in IRS-1 tyrosine phosphorylation without inhibition of insulin receptor autophosphorylation or Akt phosphorylation. A defect in this positive regulatory pathway may be a mechanism contributing to insulin resistence (11).
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