Product Pathways - PathScan ELISA
PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160
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|Kit Includes||Volume||Solution Color|
|P-Akt (S473) mAb Coated Microwells*|
|Akt1 Detection Ab||11 ml||Green|
|Anti-mouse IgG, HRP-linked Antibody||11 ml||Red|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
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, which is stored at –20°C (packaged separately).
H M R
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Introduction: CST's PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-Akt1 (Ser473) protein. Phospho-Akt (Ser473) Rabbit mAb has been coated on the microwells. After incubation with cell lysates, phospho-Akt (Ser473) protein is captured by the coated antibody. Following extensive washing, Akt1 Mouse Antibody is added to detect the captured phospho-Akt1 (Ser473) protein. HRP-linked anti-mouse antibody (#7076) is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quanitity of phospho-Akt1 (Ser473) protein.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160 detects endogenous levels of phosphorylated Akt1 at Ser473. Inhibition of phosphorylation of Akt1 (Ser473) by LY294002 in Jurkat cells is shown in Figure 1. Treatment of NIH/3T3 cells with PDGF to induce phosphorylation of Akt1 (Ser473) is also shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Figure 1: Phospho-Akt1 (Ser473) is detected in untreated Jurkat cells, where as no signal is detected with treatment with LY294002 , a specific PI3 Kinase inhibitor using PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Kit #7160.
ELISA - Western correlation
Figure 2: Treated and untreated NIH/3T3 lysates are assayed on #7160 Phospho-Akt1 (Ser 473) and #7170, Total Akt1 Sandwich ELISA kits. Total Akt1 levels can be detected in both treated and untreated lysates. Phospho-Akt1 (Ser 473) is detected only in the PDGF treated cells. The corresponding Western blots probing with #4058 and #9272 are shown in the bottom figure.
Figure 3: Relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and Absorbance at 450 nm is shown. Cells (80% confluence) were treated with PDGF (50 ng/ml) and lysed after incubation at 37ºC for 20 minutes.
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 Kip1 (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).
- 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.
- Lanner, J.T. et al. (2006) Diabetes 55, 2077-83.
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- 7252 PathScan® Phospho-Akt (Thr308) Sandwich ELISA Kit
- 7170 PathScan® Total Akt1 Sandwich ELISA Kit
- 7143 PathScan® Phospho-Akt1 (Ser473) Sandwich ELISA Antibody Pair
- 7142 PathScan® Total Akt1 Sandwich ELISA Antibody Pair
- 7144 PathScan® Phospho-Akt (Thr308) Sandwich ELISA Antibody Pair
- 4060 Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb
- 4058 Phospho-Akt (Ser473) (193H12) Rabbit mAb
- 2938 Akt1 (C73H10) Rabbit mAb
- 2967 Akt1 (2H10) Mouse mAb
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 9901 LY294002
- 7004 TMB Substrate
- 7002 STOP Solution
Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.
For Research Use Only. Not For Use In Diagnostic Procedures.