The relationship between the protein concentration of lysate from amino acid (AA)/untreated and AA/insulin-treated HEK- 293T cells and the absorbance at 450 nM using PathScan® Phospho-4E-BP1 (Thr37/Thr46) Sandwich ELISA Antibody Pair #7854 is shown. HEK-293T cells were serum-starved overnight and deprived of amino acids for 1 hour. The amino acids were replenished for 1 hour. Cells were either untreated or stimulated with 100 nM insulin for 30 minutes at 37ºC.
|REACTIVITY||H M R Mk|
|Product Includes||Volume||Cap Color|
|Phospho-4E-BP1 (Thr37/46) Capture Rabbit mAb (100X)||400 µl||Pink|
|4E-BP1 Detection Mouse mAb (100X)||400 µl||Blue|
|Anti-mouse IgG, HRP-linked Antibody (1000X)||40 µl||Yellow|
Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).
Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.
STOP Solution: (#7002)
NOTE: Reagents should be made fresh daily.
posted January 2008
revised Sepetember 2013
Protocol Id: 20
CST's PathScan® Phospho-4E-BP1 (Thr37/Thr46) Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Phospho-4E-BP1 (Thr37/Thr46) Sandwich ELISA Kit #7216. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The Phospho-4E-BP1 (Thr37/Thr46) Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a 4E-BP1 Detection Antibody and anti-Mouse IgG, HRP conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of Phospho-4E-BP1 (Thr37/Thr46) protein.
Antibodies in kit are custom formulations specific to kit.
For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Human, Mouse, Rat, Monkey
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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