Cell Signaling Technology

Product Pathways - PI3K / Akt Signaling

Akt Kinase Assay Kit (Nonradioactive) #9840

Kit Includes Quantity Source
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

Molecular Weight

27 kDa

Species Cross-Reactivity

H M R

Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

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.

Western Blotting

Western Blotting

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.
Specificity
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.

Background

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).

  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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