Product Pathways - PI3K / Akt Signaling
Akt Kinase Assay Kit (Nonradioactive) #9840
|Kinase Buffer (10X) #9802||15 ml|
|Cell Lysis Buffer (10X) #9803||15 ml|
|ATP (10 mM) #9804||50 µl|
|Anti-rabbit IgG, HRP-linked Antibody #7074||100 µl||Goat|
|20X LumiGLO® Reagent and 20X Peroxide #7003||5 ml each|
|Biotinylated Protein Ladder Detection Pack #7727||100 µl|
|Anti-biotin, HRP-linked Antibody #7075||100 µl||Goat|
|GSK-3 Fusion Protein #9237||0.04 milligrams|
|Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (Sepharose Bead Conjugate) #4070||800 µl||Rabbit|
|Phospho-GSK-3α/β (Ser21/9) (37F11) Rabbit mAb (GSK-3α Preferred) #9327||100 µl||Rabbit|
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Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Nonradioactive Akt Kinase Assay Kit provides all the reagents necessary to measure Akt kinase activity in the cell. Immobilized Akt (1G1) mAb is used to immunoprecipitate Akt from cell extracts. Then, an in vitro kinase assay is performed using GSK-3 Fusion Protein as a substrate. Phosphorylation of GSK-3 is measured by Western blotting, using Phospho-GSK-3alpha/beta (Ser21/9) Antibody.
AKT Kinase activity of PDGF-treated NIH/3T3 cell extracts was analyzed by IP/Kinase assay. Cell extracts (200 μl) were incubated overnight with Immobilized Phospho-Akt (Ser473) (D9E) Rabbit mAb #4070. After extensive washing the kinase reaction was performed in the presence of 200 μM of cold ATP and 1 μg of GSK-substrate. Phosphorylation of GSK-3 was measured by Western blot using Phospho-GSK-3 a/β (Ser21/9) Antibody #9327.
Improvements Over Conventional Assays
- No Radioactivity
- Improved sensitivity without radioactivity.
- Phospho-specific antibodies allow site-specific analysis.
- Signal to Noise
- Dramatically increased signal to noise ratio over conventional IP/kinase assays.
- Low Background Activity
- Complete System
- Includes everything needed to assay kinase activity.
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.
- Hisamoto, K. et al. (2000) Estrogen induces the Akt-dependent activation of endothelial nitric oxide synthase in vascular endothelial cells. J. Biol. Chem. 276, 3459-3467. Applications: Kinase Assay Western Blotting
- Deregibus, M. C. et al. (2003) CD40-dependent activation of phosphatidylinositol 3-kinase/Akt pathway mediates endothelial cell survival and in vitro angiogensis. J. Biol. Chem. 278, 18008-18014. Applications: Western Blotting kinase assay
- Jost, M. et al. (2001) Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway. J. Biol. Chem. 276, 6320-6326. Applications: Kinase Assay Western Blotting
- Thimmaiah, K.N. et al. (2003) Cancer Res 63, 364-74. Applications: Kinase Assay Western Blotting
- Kim, S. et al. (2004) J Biol Chem 279, 4186-95. Applications: Kinase Assay Western Blotting
- Cavin, L.G. et al. (2005) Mol Cancer Res 3, 403-12. Applications: Kinase Assay Western Blotting
- Hu, H. et al. (2005) Carcinogenesis 26, 1374-81. Applications: Kinase Assay Western Blotting
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- 9951 Wortmannin
- 9901 LY294002
- 9270 PhosphoPlus® Akt (Ser473) Antibody Kit
- 9271 Phospho-Akt (Ser473) Antibody
- 4051 Phospho-Akt (Ser473) (587F11) Mouse mAb
- 9275 Phospho-Akt (Thr308) Antibody
- 9291 Phospho-Bad (Ser112) Antibody
- 9295 Phospho-Bad (Ser136) Antibody
- 9336 Phospho-GSK-3β (Ser9) Antibody
- 3611 Phospho-Tuberin/TSC2 (Thr1462) Antibody
- 3614 Phospho-Tuberin/TSC2 (Tyr1571) Antibody
- 9272 Akt Antibody
- 7720 Prestained Protein Marker, Broad Range (Premixed Format)
- 7727 Biotinylated Protein Ladder Detection Pack
- 9315 GSK-3β (27C10) Rabbit mAb
For Research Use Only. Not For Use In Diagnostic Procedures.