Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Total Akt2 Sandwich ELISA Kit (Mouse Preferred) #7930

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
Akt Rabbit Antibody Coated Microwells 96 tests
Akt2 Mouse Detection Antibody 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

M

Reactivity Key:  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Total Akt2 Sandwich ELISA Kit (Mouse Preferred) is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Akt2 protein. An Akt rabbit antibody has been coated on the microwells. After incubation with cell lysates, Akt protein (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, Akt2 mouse antibody is added to detect captured Akt2 protein. Anti-mouse IgG, HRP-linked antibody* is then used to recognize the bound detection antibody. HRP substrate TMB is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Akt2 protein. *Antibodies in this kit are custom formulations specific to the kit.

Specificity / Sensitivity

CST's PathScan® Total Akt2 Sandwich ELISA Kit (Mouse Preferred) detects endogenous levels of Akt2 protein in mouse (NIH/3T3) cells, as shown in Figure 1. Akt protein specificity is demonstrated in Figure 2, while Akt2 kit sensitivity is shown in figure 3. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Treatment of NIH/3T3 cells with PDGF #9909 stimulates phosphorylation of Akt2 at Ser474, detected by PathScan® Phospho-Akt2 (Ser474) Sandwich ELISA Kit (Mouse Preferred) #7932, but does not affect levels of total Akt2 protein detected by PathScan® Total Akt2 Sandwich ELISA kit (Mouse Preferred) #7930. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using Akt2 (5B5) Rabbit mAb #2964 (left panel) and Phospho-Akt (Ser473) (193H12) Rabbit mAb #4058 (right panel) are shown in the bottom figure.

Isoform Specificity

Isoform Specificity

Figure 2: Demonstration of total protein sandwich ELISA kit specificity using recombinant Akt1, Akt2 and Akt3 proteins. Total Akt1 is detected by #7170 while #7930 measures endogenous levels of Akt2. Recombinant, inactive Akt protein (1.0 ng per microwell) is assayed using both ELISA kits.

Sensitivity

Sensitivity

Figure 3: The relationship between protein concentration of lysates from untreated and PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown. After starvation, NIH/3T3 cells (85% confluence) were treated with PDGF #9909 (50 ng/ml) for 10 min at 37ºC and then lysed.


Background

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).

  1. Franke, T.F. et al. (1997) Cell 88, 435-7.
  2. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  3. Franke, T.F. et al. (1995) Cell 81, 727-36.
  4. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  5. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  6. Jacinto, E. et al. (2006) Cell 127, 125-37.
  7. Cardone, M.H. et al. (1998) Science 282, 1318-21.
  8. Brunet, A. et al. (1999) Cell 96, 857-68.
  9. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  10. Cantley, L.C. and Neel, B.G. (1999) Proc Natl Acad Sci USA 96, 4240-5.
  11. Vlahos, C.J. et al. (1994) J Biol Chem 269, 5241-8.
  12. Hajduch, E. et al. (2001) FEBS Lett 492, 199-203.
  13. Cross, D.A. et al. (1995) Nature 378, 785-9.
  14. Diehl, J.A. et al. (1998) Genes Dev 12, 3499-511.
  15. Gesbert, F. et al. (2000) J Biol Chem 275, 39223-30.
  16. Zhou, B.P. et al. (2001) Nat Cell Biol 3, 245-52.
  17. Navé, B.T. et al. (1999) Biochem J 344 Pt 2, 427-31.
  18. Inoki, K. et al. (2002) Nat Cell Biol 4, 648-57.
  19. Manning, B.D. et al. (2002) Mol Cell 10, 151-62.

Application References

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