Product Pathways - Screening Technologies
PhosphoPlus® Akt (Ser473) In-Cell Duet (ICW Compatible) #7255
|Products Included||Product No.||Volume||Applicaton||Dilution||Reactivity|
|Primary Cocktail||5532||500 µl||ICW||1:10||H, M, R, Mk|
|Detection Cocktail||5531||500 µl||ICW||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|
ICW=In-Cell Western Compatible
Reactivity Key: H=Human M=Mouse R=Rat Mk=Monkey
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
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.
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.
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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LI-COR® Odyssey® are trademarks of LI-COR Biosciences.
DyLight® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
For Research Use Only. Not For Use In Diagnostic Procedures.