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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.Learn more about how we get our images.
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 datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
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.
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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