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PhosphoSitePlus® Resource

  • Additional protein information
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Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

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Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

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Western blot analysis of recombinant Akt1, Akt2 and Akt3 proteins, and extracts from various cell lines, using Akt (pan) (C67E7) Rabbit mAb.

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Western blot analysis of extracts from NIH/3T3 cells, untreated (-) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 5 min; +), and untreated (-) LNCaP and PC-3 cells, using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

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After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells.

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Immunohistochemical analysis of paraffin-embedded human melanoma using Akt (pan) (C67E7) Rabbit mAb.

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Immunoprecipitation of phospho-Akt (Thr308) from Jurkat cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Akt (pan) (C67E7) Rabbit mAb in the presence of control peptide (left) or Akt (pan) Blocking Peptide #1085 (right).

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Flow cytometric analysis of serum-starved NIH/3T3 cells, untreated (blue) or treated with Human Platelet-Derived Growth Factor AA (hPDGF-AA) #8913 (100 ng/ml, 15 min; green), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

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Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Immunohistochemical analysis using Akt (pan) (C67E7) Rabbit mAb on SignalSlide (TM) Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

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Confocal immunofluorescent analysis of C2C12 cells, insulin-treated (100 nM, 15 min; left) or treated with LY294002 #9901 (50 μM, 2 hr; right), using Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

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Flow cytometric analysis of Jurkat cells using Akt (pan) (C67E7) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

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Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Akt (pan) (C67E7) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

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Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.)

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Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Immunohistochemical analysis of frozen SKOV3 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

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Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, wortmannin #9951 and U0126 #9903 (blue), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb 4060 100 µl
Western Blotting Immunoprecipitation Immunohistochemistry Immunofluorescence Flow Cytometry
H M R Hm Mk Dm Z B 60 Rabbit IgG
Akt (pan) (C67E7) Rabbit mAb 4691 100 µl
Western Blotting Immunoprecipitation Immunohistochemistry Immunofluorescence Flow Cytometry
H M R Mk Dm 60 Rabbit IgG
Phospho-Akt (Thr308) (D25E6) XP® Rabbit mAb 13038 100 µl
Western Blotting Immunoprecipitation Immunofluorescence Flow Cytometry
H M R Mk 60 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
Western Blotting
Goat 
Anti-biotin, HRP-linked Antibody 7075 100 µl
Western Blotting
All Goat 
Biotinylated Protein Ladder Detection Pack 7727 100 µl
 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
Western Blotting
 

Product Description

Kit includes:

#13038 (100uL)

#4060 (100uL)

#4961 (100uL)

#7074 (100uL)

#7075 100ul

7003 5 ml

7727 100ul


Specificity / Sensitivity

Phospho-Akt (Ser473) detects endogenous levels of Akt only when phosphorylated at Ser473. Phospho-Akt (Thr308) detects endogenous levels of Akt1 protein only when phosphorylated at Thr308, detects endogenous levels of Akt2 protein when phosphorylated at Thr309, and detects endogenous levels of Akt3 protein when phosphorylated at Thr305. The Akt antibody detects endogenous levels of total Akt1, Akt2 and Akt3 proteins independent of their phosphorylation state. It does not cross-react with related kinases.


Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser473, Thr308, or from the carboxy-terminal sequence of mouse Akt, or residues surrounding Thr308 of human Akt1 protein.

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).


The Akt activation kit provides an economical means to evaluate the activation status of Akt at both the Ser473 and Thr308 positions as well as the total Akt protein level. The kit includes enough primary and secondary antibodies to perform ten Western mini-blot experiments.


1.  Franke, T.F. et al. (1997) Cell 88, 435-7.

2.  Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.

3.  Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.

4.  Franke, T.F. et al. (1995) Cell 81, 727-36.

5.  Cross, D.A. et al. (1995) Nature 378, 785-9.

6.  Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

7.  Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.

8.  Sarbassov, D.D. et al. (2005) Science 307, 1098-101.

9.  Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.

10.  Jacinto, E. et al. (2006) Cell 127, 125-37.

11.  Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.

12.  Cardone, M.H. et al. (1998) Science 282, 1318-21.

13.  Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.

14.  Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.

15.  Brunet, A. et al. (1999) Cell 96, 857-68.

16.  Vlahos, C. (1994) J. Biol. Chem. 269, 5241-5248.

17.  Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.

18.  Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.

19.  Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.


Entrez-Gene Id 207 , 208 , 10000
Swiss-Prot Acc. P31749 , P31751 , Q9Y243


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus is a trademark of Cell Signaling Technology, Inc.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
U.S. Patent No. 5,675,063.

9280
PhosphoPlus® Akt Activation Kit