Cell Signaling Technology

Product Pathways - Akt Signaling

Survival Marker: SignalStain® Phospho-Akt (Ser473) IHC Detection Kit #8100

Kit IncludesCap Color
Peroxidase Quench Orange
Blocking Solution Blue
Prediluted Phospho-Akt(Ser473) Antibody Purple
Prediluted Negative Control Brown
Biotinylated Secondary Antibody Green
NovaRED Substrate 1 (TM) Red
NovaRED Substrate 2 (TM) Red
NovaRED Substrate 3 (TM) Red
NovaRED Substrate 4 (TM) Red
Reagent A Gray
Reagent B Gray
Mixing Bottle Yellowish
Phospho-Akt (Ser473) Blocking Peptide # 1140 White

NovaRED™ is a trademark of Vector Laboratories.

Description

CST's Survival Marker: Signal Stain® Phospho-Akt (Ser473) IHC Detection Kit is a "ready to use" system designed to detect the activation of Akt in human tissue and cell preparations using immunohistochemistry. The kit utilizes the ABC immunoperoxidase method to detect endogenous levels of phosphorylated Akt protein. Prediluted Phospho-Akt (Ser473) Antibody is bound by a biotinylated secondary antibody. Avidin DH and biotinylated horseradish peroxidase are complexed by mixing defined amounts prior to use, and the mixture subsequently binds the secondary antibody. The macromolecular complex is localized by incubation with NovaRED™ enzyme substrate.The prediluted primary antibody, along with the ABC system, allows the user to consistently examine phosphorylated-Akt localization and offers the highest sensitivity with the lowest background.

Species Cross-Reactivity

H (M) (R)

Reactivity Key:  H=Human  M=Mouse  R=Rat
Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

Survival Marker: Signal Stain® Phospho-Akt (Ser473) IHC Detection Kit detects Akt1 only when phosphorylated at serine 473, and Akt2 and Akt3 only when phosphorylated at equivalent sites. The antibody does not detect Akt phosphorylated at other sites or related kinases such as PKC or p70 S6 kinase. This kit was developed for and is recommended for immunohistochemistry only.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues around Ser473 of mouse Akt. Antibodies are purified by protein A and peptide affinity chromatography.

IHC-P (paraffin)

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, showing cytoplasmic localization, using Survival Marker: Signal Stain® Phospho-Akt (Ser473) IHC Detection Kit (left). A serial section stained with a matched negative control demonstrates specificity of staining (right).

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 by phosphorylating and inactivating 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 due to inactivation of its effector, p70 S6 kinase and activation of 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. Nave, 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.

Application References

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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