Technical Data
A1125-20
Akt, Phosphorylated (Ser473) Survival Marker IHC Detection Kit, BioAssay™
1Kit
Kits and Assays Storage: -20°CShipping: Blue Ice
Synthetic phosphopeptide corresponding to residues around Ser473 of mouse Akt (KLH-coupled). Detects Akt1 only when phosphorylated at serine 473. Also detects Akt2 and
Akt3 only when phosphorylated at equivalent sites. Does not detect Akt phosphorylated at other
sites or related kinases such as PKC and p70 S6 kinase.

Akt, Phosphorylated (Ser473) Survival Marker IHC Detection Kit, BioAssay™ 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. 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.

Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis (1-3). This protein kinase is activated by insulin and various growth and survival factors, and functions 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 carboxyterminus at Ser473. Akt promotes cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets, including Bad (5). Forkhead transcription factors (6) and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway (7). LY294002 is a specific PI3 kinase inhibitor (8). One of the essential functions of Akt is the regulation of glycogen synthesis through phosphorylation and inactivation of glycogen synthase kinase-3a and b (9,10). Akt may also play a role in insulin stimulation of glucose transport (9). In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK3b-mediated phosphorylation and degradation of cyclin D1 (11), and by negatively regulating the cyclindependent kinase inhibitors p27 KIP (12) and p21 WAF1 (13). Akt also plays a critical role in cell growth by directly phosphorylating the mammalian target of rapamycin, mTOR (14), but more importantly through phosphorylation and inactivation of tuberin (TSC2), an mTOR inhibitor (15). 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-EP1), an inhibitor of translation (16,17).

Applications:
Suitable for use in Immunohistochemistry. Other applications not tested.

Purity (antibodies): Purified by Protein A and immunoaffinity chromatography.

Quantitity: Sufficient material for 150 slides.

Kit Components: Cap Color
Peroxidase Quench Orange
Blocking Solution Blue
Prediluted Phospho-Akt (Ser473) Antibody Purple
Prediluted Negative Control Brown
Biotinylated Secondary Antibody Green
A and B Reagents Gray
NovaRed™ Substrate* Red
Phospho-Akt (Ser473) Blocking Peptide White
* NovaRed™ is a trademark of Vector Labs

Components are ready to use and should not be aliquotted.

Blocking Solution, Prediluted Phospho-Akt (Ser473) Antibody, Prediluted Negative Control and
Biotinylated Secondary Antibody contain 0.05% sodium azide.

Storage and Stability:
Storage: Store at 4°C.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Application References: 1. Gupta, A.K., et al., Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin. Cancer Res. 8: 885-892 (2002). Applications: IHC (paraffin). 2. Malik, S.N., et al., Immunohistochemical demonstration pf phospho-Akt in high gleason grade prostate cancer. Clin. Cancer Res. 8(4): 1168-1171 (2002). Applications: IHC (paraffin). 3. Noshita, N., et al., Akt phosphorylation and neuronal survival after traumatic brain injury in mice. Neurobiol. Dis. 9: 294-304 (2002). Applications: IHC (paraffin). 4. Roy, H.K., et al., AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis 23: 201-205 (2002). Applications: IHC (paraffin). Background References: 1. Franke, T.F., Cell 88: 435-437 (1997). 2. Burgering, B.T., Coffer, P.J., Nature 376: 599-602 (1995). 3. Franke, T.F., et al., Cell 81: 727-736 (1995). 4. Alessi, D.R., et al., EMBO J. 15: 6541-6551 (1996). .) Cardone, M.H., et al., Science 282: 1318-1321 (1998). 6. Brunet, A., et al., Cell 96: 857-868 (1999). 7. Cantley, L.C., et al., PNAS USA 96: 4240-4245 (1999). 8. Vlahos, C., et al.,J. Biol. Chem. 269: 5241-5248 (1994) . 9. Hajduch, E., et al., FEBS Lett. 492: 199-203 (2000). 10. Cross, D.A., et al., Nature 373: 785-789 (1995). 11. Diehl, et al., Genes Dev. 12: 3499-3511 (1998). 12. Gesbert, F., et al., J. Biol. Chem. 273: 39223-39230 (2000). 13. Zhou, B.P. et al., Nat. Cell Biol. 3: 245-252 (2001). 14. Nave, B.T., et al., Biochem. J. 344: 427-431 (1999). 15. Manning, B.D., et al., Mol. Cell 4: 648-657 (2000). 16. Manning, B.D., et al., Mol. Cell 10: 151-162 (2002). 17. Inoki, K., et al., Nat. Cell Biol. 4: 648-657 (2002).

Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.