PathScan^® Phospho-Akt2 (Ser474) Sandwich ELISA Kit (Mouse Preferred) #7932

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	Catalog #		Size & Concentration		Price(£)		Qty
	7932S		1 Kit (96 assays)		377.00
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Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).

Species Cross-Reactivity

M

Reactivity Key:  M=Mouse

Description

CST's PathScan^® Phospho-Akt2 (Ser474) Sandwich ELISA Kit (Mouse Preferred) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Akt2 protein phosphorylated at Ser474. A phospho-Akt rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Akt protein is captured by coated antibody. Following extensive washing, a biotinylated Akt2 rabbit antibody is added to detect the captured phospho-Akt2 protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Akt2 phosphorylated at Ser474.

Specificity / Sensitivity

CST's PathScan^® Phospho-Akt2 (Ser474) Sandwich ELISA Kit (Mouse Preferred) detects endogenous levels of Akt2 protein phosphorylated at Ser474 in mouse (NIH/3T3) cells, as shown in Figure 1. Phospho-Akt protein specificity is demonstrated in Figure 2, while Akt2 kit sensitivity is shown in figure 3.

ELISA - Western correlation

Figure 1: Treatment of NIH/3T3 cells with PDGF #9909 stimulates phosphorylation of Akt2 at Ser474, detected by PathScan^® Phospho-Akt2 (Ser474) Sandwich ELISA Kit (Mouse Preferred) #7932, but does not affect levels of total Akt2 protein detected by PathScan^® Total Akt2 Sandwich ELISA kit (Mouse Preferred) #7930. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Akt2 (5B5) Rabbit mAb #2964 (left panel) and Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058 (right panel) are shown in the bottom figure.

Isoform Specificity

Figure 2: Demonstration of phospho-Akt protein sandwich ELISA kit specificity using recombinant phosphorylated Akt1, Akt2 and Akt3 proteins. Phospho-Akt1 (Ser473) is detected by #7160 while #7932 measures levels of phospho-Akt2 (Ser474). Recombinant phosphorylated Akt protein (1.0 ng per microwell) is assayed using both ELISA kits.

Sensitivity

Figure 3: The relationship between protein concentration of lysates from untreated or PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with PDGF #9909 (50 ng/ml) for 10 min at 37ºC and then lysed.

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).

1. Franke, T.F. et al. (1997) Cell 88, 435-7.
2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
3. Franke, T.F. et al. (1995) Cell 81, 727-36.
4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
6. Jacinto, E. et al. (2006) Cell 127, 125-37.
7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
8. Brunet, A. et al. (1999) Cell 96, 857-68.
9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
13. Cross, D.A. et al. (1995) Nature 378, 785-9.
14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
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.

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7932:

Sandwich ELISA

• 7930 PathScan^® Total Akt2 Sandwich ELISA Kit (Mouse Preferred)
• 4057 Akt2 (54G8) Rabbit mAb (IHC Specific)
• 2964 Akt2 (5B5) Rabbit mAb
• 3063 Akt2 (D6G4) Rabbit mAb
• 9803 Cell Lysis Buffer (10X)
• 9808 Phosphate Buffered Saline (PBS-20X)
• 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
• 9998 BSA
• 7004 TMB Substrate
• 7002 STOP Solution

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.