Revision 1

#7239Store at +4C

 

()

Species Cross Reactivity

H M

UniProt ID:

#P27361, #P62753, #P28482, #P31749

Entrez-Gene Id:

#5595, #6194, #5594, #207

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Color Storage Temp
Cell Growth Multi-Target 66422 96 tests +4C
TMB Substrate 7004 11 ml Colorless +4C
STOP Solution 7002 11 ml Colorless +4C
Sealing Tape 54503 2 ea +4C
ELISA Wash Buffer (20X) 9801 25 ml Colorless +4C
ELISA Sample Diluent 11083 25 ml Blue +4C
Cell Lysis Buffer (10X) 9803 15 ml Yellowish -20C

*The microwell plate is supplied as 12 8-well modules - Each module is designed to break apart for 8 tests.

Description

CST’s PathScan® Cell Growth Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204). These molecules represent key signaling proteins in pathways controlling growth and differentiation. Sixteen assays are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual sandwich ELISA kits*. Briefly, a capture antibody** has been coated onto the microwells. After incubation with cell lysates, the target protein is captured by the coated antibody. Following extensive washing, a detection antibody** is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein. * See companion products. ** Antibodies in kit are custom formulations specific to kit.

Specificity/Sensitivity

CST's PathScan® Cell Growth Multi-Target Sandwich ELISA Kit #7239 detects endogenous levels of six proteins: S6 ribosomal protein, phospho-S6 ribosomal protein (Ser235/236), Akt1, phospho-Akt (Ser473), phosho-Akt (Thr308) and phospho-p44/42 MAPK (Thr202/Tyr204). Activation of these proteins can be observed over time in response to PDGF. As shown in Figure 1, stimulation of serum-starved NIH/3T3 cells with PDGF promotes phosphorylation of Akt1 at Thr308 and Ser473, S6 ribosomal protein at Ser235/236 and p44/42 MAPK at Thr202/Tyr204. The level of each target protein (phospho and nonphospho) remains unchanged throughout the 80 minute time course as demonstrated by Western analysis.
The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits*. *See companion products.
This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Background

Akt is a protooncogene with a critical regulatory role in diverse cellular processes including growth, survival and the cell cycle. Akt is also a major regulator of insulin signaling and glucose metabolism (1-4). Akt is activated by PI3 kinase signaling and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473 by the mTOR-rictor complex (TORC1) (5-7).
Both p44 and p42 MAP kinases (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation (8-13). MAP kinases are activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. Activation of MAP kinases occurs through phosphorylation of threonine and tyrosine (202 and 204 of human MAP kinase or 183 and 185 of rat MAP kinase) at the sequence T*EY* by a single upstream MAP kinase kinase (MEK) (14,15).
To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (16,17). Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylates the S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5' untranslated regions (17). This group of mRNAs (5'TOP) encodes proteins involved in cell cycle progression and proteins that are part of the translational machinery, such as ribosomal proteins and elongation factors (17,18). The main in vivo S6 ribosomal protein phosphorylation sites, including Ser235, Ser236, Ser240 and Ser244, are located within a small 19 amino acid region in the S6 carboxy terminus (19,20).

  1. Kim, D. and Chung, J. (2002) J Biochem Mol Biol 35, 106-15.
  2. Zdychová, J. and Komers, R. (2005) Physiol Res 54, 1-16.
  3. Song, G. et al. J Cell Mol Med 9, 59-71.
  4. Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.
  5. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  6. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  7. Jacinto, E. et al. (2006) Cell 127, 125-37.
  8. McKay, M.M. and Morrison, D.K. (2007) Oncogene 26, 3113-21.
  9. Pearson, G. et al. (2001) Endocr Rev 22, 153-83.
  10. Marshall, C.J. (1995) Cell 80, 179-85.
  11. Hunter, T. (1995) Cell 80, 225-36.
  12. Hill, C.S. and Treisman, R. (1995) Cell 80, 199-211.
  13. Cowley, S. et al. (1994) Cell 77, 841-52.
  14. Sturgill, T.W. et al. (1988) Nature 334, 715-8.
  15. Payne, D.M. et al. (1991) EMBO J 10, 885-92.
  16. Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.
  17. Peterson, R.T. and Schreiber, S.L. (1998) Curr Biol 8, R248-50.
  18. Jefferies, H.B. et al. (1997) EMBO J 16, 3693-704.
  19. Ferrari, S. et al. (1991) J Biol Chem 266, 22770-5.
  20. Flotow, H. and Thomas, G. (1992) J Biol Chem 267, 3074-8.

Background References

    Cross-Reactivity Key

    H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    PathScan is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.