Western blot analysis of extracts from NIH/3T3 cells, untreated, PDGF-treated (50 ng/ml), PDGF and wortmannin-treated (200 nM) or PDGF and rapamycin-treated (5 nM), and MCF-7 cells, untreated or IGF-1 treated for the indicated times, using Phospho-Tuberin/TSC2 (Thr1462) Antibody (upper) or Tuberin/TSC2 Antibody #3612 (lower).
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 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
Phospho-Tuberin/TSC2 (Thr1462) Antibody detects endogenous levels of tuberin only when phosphorylated at threonine 1462. This antibody does not detect tuberin phosphorylated at other sites.Species Reactivity:
Human, MouseSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr1462 of human tuberin. Antibodies are purified by protein A and peptide affinity chromatography.
Tuberin is a product of the TSC2 tumor suppressor gene and an important regulator of cell proliferation and tumor development (1). Mutations in either TSC2 or the related TSC1 (hamartin) gene cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by development of multiple, widespread non-malignant tumors (2). Tuberin is directly phosphorylated at Thr1462 by Akt/PKB (3). Phosphorylation at Thr1462 and Tyr1571 regulates tuberin-hamartin complexes and tuberin activity (3-5). In addition, tuberin inhibits the mammalian target of rapamycin (mTOR), which promotes inhibition of p70 S6 kinase, activation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translation initiation), and eventual inhibition of translation (3,6,7).
Tuberin is phosphorylated on Ser939 and Thr1462 in response to PI3K activation, and that the human TSC complex is a direct biochemical target of the PI3K/Akt pathway (3). This data complements Drosophila genetics studies suggesting the possible involvement of the tuberin-hamartin complex in the PI3K/Akt mediated insulin pathway (8-10).
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