Akt, also referred to as PKB or Rac, plays a critical role in controlling the balance between survival and apoptosis. This protein kinase is activated by insulin and various growth and survival factors, and functions in a wortmannin-sensitive pathway involving PI3 kinase. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1, and by phosphorylation within the carboxy-terminus at Ser473. Akt promotes cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets, including Bad, Forkhead transcription factors and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/Akt signaling pathway. LY294002 is a specific PI3 kinase inhibitor. One of the essential functions of Akt is the regulation of glycogen synthesis through phosphorylation and inactivation of glycogen synthase kinase-3 alpha and beta. Akt may also play a role in insulin stimulation of glucose transport.

In addition to its role in survival and glycogen synthesis, Akt is involved in cell cycle regulation by preventing GSK3 beta-mediated phosphorylation and degradation of cyclin D1, and by negatively regulating the cyclin-dependent kinase inhibitors p27 KIP and p21 WAF1. Akt also plays a critical role in cell growth by directly phosphorylating the mammalian target of rapamycin, mTOR, but more importantly through phosphorylation and in activation of tuberin (TSC2), an mTOR inhibitor. Inhibition of mTOR stops the protein synthesis machinery due to inactivation of its effec-tor, p70 S6 kinase, and activation of the eukaryotic initiation factor, 4E binding protein 1 (4E-EP1), an inhibitor of translation.

Akt1 shares 81% and 83% amino acid identity with Akt2 andAkt3, respectively.Although the three isoforms show broad tissue distribution, Akt1 is the most ubiquitously expressed. Akt2 is expressed at a lower level than Akt1 except in insulin-responsive tissues where it predominates. In particular, Akt2 is more abundant and more highly activated than Akt1 in adipocytes. Akt3 is expressed at the lowest level except in testes and brain. The C-terminal sequence of the three isoforms are shown below. Note that the phosphorylation site slightly differs between the three isoforms.Thus the phosphorylation site Ser⁴⁷³ of Akt1 corresponds to Ser⁴⁷⁴ in Akt2 and Ser⁴⁷² in Akt3 respectively.

Akt1: V⁴⁶¹DSERRPHFPQFS473YSASSTA⁴⁸⁰

Akt2: L⁴⁶¹LELDQRTHFPQFS474YSASIRE⁴⁸¹

Akt3: D⁴⁶¹NERRPHFPQFS472YSASGRE⁴⁷⁹

Specificity/Sensitivity:

Phospho-Akt (Ser473) Antibody detects endogenous levels of Akt1 only when phosphorylated at serine 473. This antibody also recognizes Akt2 and Akt3 when phosphorylated at the corresponding residues. It does not recognize Akt phos-phorylated at other sites, nor does it recognize phos-phorylated forms of related kinases such as PKC or p70 S6 kinase.

Source/Purification:

Monoclonal antibodies are produced by immunizing rabbits with a synthetic phosphopeptide (KLH coupled) corresponding to residues around Ser473 of mouse Akt.

Storage and Stability:

Each vial contains 100ul affinity purified rabbit anti-phospho-Akt antibody in a special stabilizer. Real time studies that this special stabilizer helped retain most of antibody activity for 24 months while store at 4℃.

Recommended Antibody Dilutions: