Western blot analysis of extracts from NIH/3T3 cells, serum-starved for 14 hours and then either left untreated or treated with insulin (150 nM) for 15 minutes, using Phospho-PRAS40 (Thr246) Antibody.
Western blot analysis of extracts from NIH/3T3 cells, serum-starved for 14 hours and then either left untreated or treated with insulin (150 nM) for 15 minutes, using Phospho-PRAS40 (Thr246) Antibody. The experiment was performed in the presence of phospho-peptide specific to phospho-PRAS40 (Thr246) (upper panel), the corresponding nonphospho-peptide (middle panel) and in the absence of any blocking peptide (lower panel).
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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-PRAS40 (Thr246) Antibody detects endogenous levels of PRAS40 protein only when phosphorylated at Thr246.
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to the sequence surrounding Thr246 of human PRAS40. Antibodies are purified by peptide affinity chromatography.
Many growth factors and hormones induce the phosphoinositide 3-kinase signaling pathway, which results in the activation of downstream effector proteins such as the serine/threonine kinase Akt (1,2). One known Akt substrate is a 40 kDa, proline-rich protein (PRAS40) that binds to 14-3-3 proteins (2). PRAS40 also binds mTOR to transduce Akt signals to the mTOR complex. Inhibition of mTOR signaling stimulates PRAS40 binding to mTOR, which in turn inhibits mTOR activity (3). PRAS40 interacts with raptor in mTOR complex 1 (mTORC1) in insulin-deprived cells and inhibits the activation of the mTORC1 pathway mediated by the cell cycle protein Rheb. Phosphorylation of PRAS40 by Akt at Thr246 relieves PRAS40 inhibition of mTORC1 (4). mTORC1 in turn phosphorylates PRAS40 at Ser183 (5).
Explore pathways + proteins related to this product.
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