| 货号 | 7170C |
| 描述 | CSTs PathScan® Total Akt1 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Akt1 protein. An Akt Antibody has been coated onto the microwells. After incubation with cell lysates, the Akt protein is captured by the coated antibody. Following extensive washing, Akt1 Mouse Monoclonal Antibody is added to detect the captured total Akt1 protein. HRP-linked anti-mouse antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Akt1 protein. |
| 反应种属 | Human/Mouse |
| 应用 | ELISA |
| 目标/特异性 | CSTs PathScan® Total Akt1 Sandwich ELISA Kit detects endogenous levels of Akt1 including phosphorylated and nonphosphorylated. Inhibition of phosphorylation of Akt at serine 473 by LY294002 in NIH/3T3 cells is detected by PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit , #7160, while the level of total Akt1 protein detected by this ELISA kit #7170 is unchanged, as seen in Figure 1. Similar results have also observed in Jurkat cells (data not shown). This ELISA kit does not detect related kinases, such as PKC and p70 S6 kinase. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species. |
| 供应商 | CST |
| 背景 | 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 (18,19). |
| 存放说明 | 4C |
| 参考文献 | Franke, T.F. et al. (1997) Cell 88, 435-7. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602. Franke, T.F. et al. (1995) Cell 81, 727-36. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51. Sarbassov, D.D. et al. (2005) Science 307, 1098-101. Jacinto, E. et al. (2006) Cell 127, 125-37. Cardone, M.H. et al. (1998) Science 282, 1318-21. Brunet, A. et al. (1999) Cell 96, 857-68. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203. Cross, D.A. et al. (1995) Nature 378, 785-9. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57. Manning, B.D. et al. (2002) Mol Cell 10, 151-62. Harris, S.L. et al. (2005) Proc Natl Acad Sci U S A 102, 16297-302. |
Figure 1: Treatment of NIH/3T3 cells with various concentrations of LY294002 blocks PDGF-stimulated phosphorylation of Akt1 at serine 473 detected by PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit , #7160, but does not affect the level of total Akt1 protein detected by PathScan Total Akt1 Sandwich ELISA kit #7170. OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-Akt (Ser473) Antibody #9271 or Akt Antibody #9272, is shown in the bottom figure. Figure 1 NIH/3T3细胞采用不同浓度LY294002阻断PDGF诱导的Akt1 在Ser473位点的磷酸化，检测用PathScan Phospho-Akt1 (Ser473) Sandwich ELISA kit , #7160。但不影响Akt1总蛋白的水平，检测用PathScan Total Akt1 Sandwich ELISA kit #7170。在450nm处读取吸光度值（上图），相对应western blot采用抗体为Phospho-Akt (Ser473) Antibody #9271 或Akt Antibody #9272，见下图。 | |
