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7255S 1 Kit (1 X 96 well plate)
7255L 1 Kit (5 X 96 well plates)

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Analysis of MDA-MB-468 cells exposed to varying concentrations of LY294002 (PI3 Kinase Inhibitor) #9901 for 2 hours, followed by hEGF #8916 stimulation for 20 minutes. The phosphorylation status of Akt, as well as the total protein expression level, was measured simultaneously using the PhosphoPlus® Akt (Ser473) In-Cell Duet (ICW Compatible) #7255. With increasing concentrations of LY294002, a significant decrease (~3-fold) in phospho-Akt signal as compared to the hEGF-stimulated control was observed, while total Akt protein levels remained unchanged and were used to normalize the data. When using phospho-Akt as a measurement, the IC50 of this compound was 2.7 μM. Data and images were generated using the LI-COR® Biosciences Odyssey® Infrared Imaging System.

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Kit Includes

Products Included No. Volume Applicaton Dilution Reactivity
Primary Cocktail 5532 500 µl In-Cell Western Compatible 1:10 Human
Mouse
Rat
Monkey
Detection Cocktail 5531 500 µl In-Cell Western Compatible 1:10 N/A
Kit Analytes Detection Dye Ex(max) (nm) Em(max) (nm)
Phospho-Akt (Ser473) DyLight® 800 777 794
Total Akt DyLight® 680 692 712

Product Description

PhosphoPlus® Akt (Ser473) In-Cell Duet from Cell Signaling Technology (CST) provides an easy method to assess protein activation status using a multi-well plate scanner with near infrared detection capabilities, such as the LI-COR® Biosciences Odyssey® Infrared Imaging System. This kit contains a pre-optimized activation-state and total protein antibody cocktail, selected based on superior performance. Phosphorylated and total protein are detected simultaneously in the same well, allowing levels of phosphorylated protein to be normalized to total protein. A near infrared detection cocktail is also included.


Specificity / Sensitivity

Phospho-Akt (Ser473) antibody detects endogenous levels of Akt only when phosphorylated at Ser473. Total Akt detects endogenous levels of total Akt protein. This antibody does not cross-react with other related proteins.


Species Reactivity: Human

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Ser473 of human Akt or at the carboxy terminal sequence of human Akt.

Akt, also referred to as PKB or Rac, plays a critical role in controlling survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (2,3). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (4) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (5,6). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including Bad (7), forkhead transcription factors (8), c-Raf (9), and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (10). LY294002 is a specific PI3 kinase inhibitor (11). Another essential Akt function is the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (12,13). Akt may also play a role in insulin stimulation of glucose transport (12). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK-3β mediated phosphorylation and degradation of cyclin D1 (14) and by negatively regulating the cyclin dependent kinase inhibitors p27 Kip (15) and p21 Waf1/CIP1 (16). Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor (17). More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex (18). Inhibition of mTOR stops the protein synthesis machinery by inactivating p70 S6 kinase and activating the eukaryotic initiation factor 4E binding protein 1 (4E-BP1), an inhibitor of translation (18,19).


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8.  Jacinto, E. et al. (2006) Cell 127, 125-37.

9.  Cardone, M.H. et al. (1998) Science 282, 1318-21.

10.  Brunet, A. et al. (1999) Cell 96, 857-68.

11.  Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.

12.  Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.

13.  Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.

14.  Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.

15.  Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.

16.  Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.

17.  Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.

18.  Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.

19.  Manning, B.D. et al. (2002) Mol Cell 10, 151-62.


Entrez-Gene Id 207
Swiss-Prot Acc. P31749

Protein Specific References

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Misra UK and Pizzo SV (2012) J Cell Biochem 113, 1488–500


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
LI-COR® is a registered trademark of LI-COR, Inc.
Odyssey® is a registered trademark of LI-COR, Inc.