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  3. PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit

PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272

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Inquiry Info. # 7272

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    Supporting Data

    REACTIVITY H M
    Application Key:
    • ELISA-ELISA 
    Species Cross-Reactivity Key:
    • H-Human 
    • M-Mouse 

    Product Information

    Product Description

    CST's PathScan® Signaling Nodes 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 Akt1, phospho-Akt1 (Ser473), phospho-MEK1 (Ser217/221), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-NF-κB p65 (Ser536). These molecules represent convergence points and key regulatory proteins in signaling pathways controlling cellular events such as growth and differentiation, energy homeostasis, and the response to stress and inflammation. Sixteen tests are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual PathScan® 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.
    Antibodies in kit are custom formulations specific to kit.
    *See companion products.

    Protocol

    Specificity / Sensitivity

    CST's PathScan® Signaling Nodes Multi-Target Sandwich ELISA Kit #7272 detects endogenous levels of six proteins: Akt1, phospho-Akt1 (Ser473), phospho-MEK1 (Ser217/221), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-NF-κB p65 (Ser536). Differential phosphorylation of these proteins can be observed over time in response to various growth factor and cytokine treatments, as shown in Figure 1.
    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.

    Species Reactivity:

    Human, Mouse

    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 withing the carboxyl terminus at Ser473 by PDK2 (5-7).
    MEK1 and MEK2 are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling cell growth and differentiation. Activation of MEK1 and MEK2 occurs through phosphorylation of serine 217 and serine 221 by Raf-like molecules. MEK activates p44 and p42 MAP kinase (8-10).
    p38 MAP kinase (MAPK) participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokines and a variety of cellular stresses. MKK3, MKK6 and SEK (MKK4) activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182 (11-14).
    The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors. Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation and DNA binding (15,16).
    Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammation, stress and immune responses. There are five family members in mammals: RelA/p65, c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). These proteins function as dimeric transcription factors. In unstimulated cells, NF-κB/Rel proteins are sequestered in the cytoplasm and inhibited by the IκB proteins. NF-κB-activating agents induce phosphorylation of IκB's, targeting them for degradation and thereby releasing the NF-κB/Rel complexes. Active NF-κB/Rel complexes are further activated by phosphorylation (17-20).
    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. Alessi, D.R. et al. (1994) EMBO J 13, 1610-9.
    9. McKay, M.M. and Morrison, D.K. (2007) Oncogene 26, 3113-21.
    10. Pearson, G. et al. (2001) Endocr Rev 22, 153-83.
    11. Raingeaud, J. et al. (1995) J Biol Chem 270, 7420-6.
    12. Raman, M. et al. (2007) Oncogene 26, 3100-12.
    13. Zarubin, T. and Han, J. (2005) Cell Res 15, 11-18.
    14. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
    15. O'Shea, J.J. et al. (2002) Cell 109 Suppl, S121-31.
    16. Kaptein, A. et al. (1996) J Biol Chem 271, 5961-4.
    17. Ghosh, S. and Karin, M. (2002) Cell 109 Suppl, S81-96.
    18. DiDonato, J. et al. (1996) Mol Cell Biol 16, 1295-304.
    19. Sakurai, H. et al. (1999) J Biol Chem 274, 30353-6.
    20. Mattioli, I. et al. (2004) J Immunol 172, 6336-44.

    Database Links

    UniProt ID: Q16539 , P40763 , Q04206 , P31749 , Q02750


    Entrez-Gene Id: 1432 , 6774 , 5970 , 207 , 5604


    Limited Uses

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    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.

    For Research Use Only. Not For Use In Diagnostic Procedures.
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