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Each lot of this antibody is quality control tested by Western blotting. Western blotting suggested working dilution(s): Use 10 µg antibody per 5 ml antibody dilution buffer for each mini-gel. It is recommended that the reagent be titrated for optimal performance for each application.
COA:
Application Notes:
The optimal concentration should be determined by titration for each individual assay of interest.
Panel A: Total cell lysates from untreated 3T3 cells (Lane 1) or PDGF activated 3T3 cells (Lane 3) were resloved by electrophoreis, transferred to nitrocellulose and probed with anti-phosphor-Akt (S473) antibody. Proteins were visualized by HRP-conjugated anti-rabbit secondary antibody and a chemiluminescence system. Panel B: Same lysates were used as panel A. The membrane was probed with anti-phosphor-Akt (S473) plus phosphor-peptide used to generate the antibody. This panel shows that phosphor-peptide can specifically block the phosphor-Akt antibody. Panel C: Same lysates were used as panel A. The membrane was probed with anti-phosphor-Akt (S473) plus non-phosphor-peptide. This panel shows that non-phosphor-peptide cannot block the phosphor-signal. Panel D: The same membranes from panel A, B, and C were stripped and re-probed with anti-b-actin antibody.
Akt (also known as protein kinase B alpha) is a 60 kD serine/threonine specific kinase containing a pleckstrin domain. Akt plays a critical role in controlling survival and apoptosis. This kinase is ubiquitously expressed and translocates to the membrane upon activation. This protein kinase is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI 3-kinase. 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 a mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1. Activated Akt can then phosphorylate a wide range of substrates including transcription factors (e.g. FOXO1), kinases (GSK-3,Raf-1, ASK, Chk1) and other proteins with important signaling roles (e.g. Bad, MDM2). Akt promotes cell survival by inhibiting apoptosis through phosphorylation and by inactivation of several targets, including Bad, forkhead transcription factor, c-Raf and caspase-9. Akt also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor. More importantly, Akt phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex. The phosphor-Akt (S473) antibody specifically detects phosphorlated Akt at the Ser473 site.
Other Names:
Protein kinase B alpha (PRK-BA), Serine/Threonine specific kinase RAC alpha, Protein kinase Akt
Structure:
Serine/Threonine family of kinases, pleckstrin domain; 60 kD
Distribution:
Ubiquitously expressed, translocates to the membrane upon activation.
Function:
Catalytically inactive multimeric complex. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473 by PDK2.
Interaction:
Interacts through pleckstrin homology domain with second messengers. Interacts with AKTIP, CDKN1B.
Cellular Sources:
Phosphorylates substrates in cytoplasm, nucleus.
Antigen References:
1. Jacinto E, et al. 2006. Cell 127:125. 2. Inoki K, et al. 2002. Nat. Cell. Biol. 4:648. 3. Franke TF, et al. 1995. Cell 81:727. 4. Brunet A, et al. 1999. Cell 96:857. 5. Yang WL, et al. 2010 Cell Cycle 9:487.
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This data display is provided for general comparisons between formats. Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors. If you need assistance with selecting the best format contact our expert technical support team.
Purified anti-Akt Phospho (Ser473)
Panel A: Total cell lysates from untreated 3T3 cells (Lane 1) or PDGF activated 3T3 cells (Lane 3) were resloved by electrophoreis, transferred to nitrocellulose and probed with anti-phosphor-Akt (S473) antibody. Proteins were visualized by HRP-conjugated anti-rabbit secondary antibody and a chemiluminescence system. Panel B: Same lysates were used as panel A. The membrane was probed with anti-phosphor-Akt (S473) plus phosphor-peptide used to generate the antibody. This panel shows that phosphor-peptide can specifically block the phosphor-Akt antibody. Panel C: Same lysates were used as panel A. The membrane was probed with anti-phosphor-Akt (S473) plus non-phosphor-peptide. This panel shows that non-phosphor-peptide cannot block the phosphor-signal. Panel D: The same membranes from panel A, B, and C were stripped and re-probed with anti-b-actin antibody.
