Western blot analysis of extracts from HeLa cells, serum-starved and stimulated with insulin (100 nM for 5 minutes), with (+) or without (-) λ phosphatase treatment, using Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (Biotinylated) (upper) and 4E-BP1 (53H11) Rabbit mAb #9644 (lower).
|REACTIVITY||H M R Mk Dm|
|MW (kDa)||15 to 20|
This Cell Signaling Technology (CST) antibody is conjugated to biotin under optimal conditions. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb #2855.
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
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.
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 266
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb detects endogenous levels of 4E-BP1 only when phosphorylated at Thr37 and/or Thr46. This antibody may cross-react with 4E-BP2 and 4E-BP3 when phosphorylated at analagous sites.
Human, Mouse, Rat, Monkey, D. melanogaster
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr37 and Thr46 of mouse 4E-BP1.
Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent translation by binding to the translation initiation factor eIF4E. Hyperphosphorylation of 4E-BP1 disrupts this interaction and results in activation of cap-dependent translation (1). Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase regulate 4E-BP1 activity (2,3). Multiple 4E-BP1 residues are phosphorylated in vivo (4). While phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation at Ser65 and Thr70 (5).
Explore pathways + proteins related to this product.
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