This peptide is used to block Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058, Phospho-Akt (Ser473) (736E11) Rabbit mAb (IHC Specific) #3787 and Phospho-Akt (Ser473) (D9E) Rabbit mAb #4060 reactivity.
The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks Phospho-Akt (Ser473) Rabbit mAb #4060 and (IHC Specific) #3787 signal completely in immunohistochemistry and Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058 signal in flow cytometry.
Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry and flow cytometry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 µl. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. For flow cytometry, add antibody at the recommended dilution to 100 µl incubation buffer. Next, add 10 µl blocking peptide. Incubate at room temperature for 15-30 minutes before applying to cells. *Recommended antibody dilutions can be found on the antibody datasheet.
Supplied in 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA, 5% glycerol and 1% DMSO. Store at –20°C.
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).
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