Western blot analysis of cell extracts from CHO cells overexpressing insulin receptor and IRS-1, untreated or treated with insulin, using Phospho-IRS-1 (Ser332/336) Antibody (upper and middle) or IRS-1 Antibody #2382 (lower). The middle blot was treated with calf intestinal phosphatase (CIP) before antibody probing.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 (Ser332/336) Antibody detects transfected levels of IRS-1 when phosphorylated at Ser332/336. It also detects IRS-1 protein when singly phosphorylated at Ser332 or Ser336 of human IRS-1. This antibody does not cross-react with other related phosphoproteins.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser332/336 of mouse IRS-1 (equivalent to Ser337/341 of human IRS-1). Antibodies are purified by 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).
GSK-3-mediated IRS-1 serine phosphorylation leads to inhibition of insulin-stimulated IRS-1 signaling. Ser332 and Ser336 of IRS-1 are situated in a glycogen synthase kinase-3 (GSK-3) consensus motif (SXXXS), and it has been shown that Ser332 is the actual GSK-3 phosphorylation site while Ser336 provides a " priming" site necessary for GSK-3 action (11).
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|2580S||100 µl (10 western blots)||$ 297.0|