For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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: 263
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr642 of human AS160 protein.
Insulin is a major hormone controlling critical energy functions, such as glucose and lipid metabolism. Insulin binds to and activates the insulin receptor (IR) tyrosine kinase, which phosphorylates and recruits adaptor proteins. The signaling pathway initiated by insulin and its receptor stimulates glucose uptake in muscle cells and adipocytes through translocation of the Glut4 glucose transporter from the cytoplasm to the plasma membrane (1). A 160 kDa substrate of the Akt Ser/Thr kinase (AS160, TBC1D4) is a Rab GTPase-activating protein that regulates insulin-stimulated Glut4 trafficking. AS160 is expressed in many tissues including brain, kidney, liver, and brown and white fat (2). Multiple Akt phosphorylation sites have been identified on AS160 in vivo, with five sites (Ser318, Ser570, Ser588, Thr642, and Thr751) showing increased phosphorylation following insulin treatment (2,3). Studies using recombinant AS160 demonstrate that insulin-stimulated phosphorylation of AS160 is a crucial step in Glut4 translocation (3) and is reduced in some patients with type 2 diabetes (4). The interaction of 14-3-3 regulatory proteins with AS160 phosphorylated at Thr642 is a necessary step for Glut4 translocation (5). Phosphorylation of AS160 by AMPK is involved in the regulation of contraction-stimulated Glut4 translocation (6).
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