Product Pathways - MAPK Signaling
p44/42 MAP Kinase Assay Kit (Nonradioactive) #9800
|9800||carrier free and custom formulation / quantity||email request|
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|Human, Mouse, Rat|
|Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (Sepharose Bead Conjugate) #3510||800 µl||Rabbit|
|Phospho-Elk-1 (Ser383) (2B1) Mouse mAb #9186||100 µl||Mouse|
|Elk-1 Fusion Protein #9184||40 µl|
|Kinase Buffer (10X) #9802||15 ml|
|Cell Lysis Buffer (10X) #9803||15 ml|
|ATP (10 mM) #9804||50 µl|
|Anti-mouse IgG, HRP-linked Antibody #7076||100 µl||Horse|
|Anti-biotin, HRP-linked Antibody #7075||100 µl||Goat|
|Biotinylated Protein Ladder Detection Pack #7727||100 µl|
|20X LumiGLO® Reagent and 20X Peroxide #7003||5 ml each|
Nonradioactive p44/42 MAP Kinase Assay Kit provides all the reagents necessary for measuring p44/42 MAP kinase activity in the cell. Immobilized Phospho-p44/42 MAPK (Thr202/Tyr204) mAb is used to immunoprecipitate active p44/42 MAP kinase from cell extracts, then an in vitro kinase assay is performed using Elk-1 protein as a substrate. Elk-1 phosphorylation is then detected by Western blotting using Phospho-Elk-1 (Ser383) Antibody.
p44/42 MAP Kinase-induced phosphorylation of Elk-1 was measured by immunoblotting with Phospho-Elk-1 (Ser 383)9186, mouse mAb. Lysate titrations were run by immunoprecipitating various amounts of Phospho-p44/42 from 3T3, PDGF lysates and 3T3, U0126 lysates. Phosphorylation of Elk-1 (Ser383) is clearly seen in the positive control lysate compared with the same amounts of the negative control.
Improvements Over Conventional Assays
- No Radioactivity
- Improved sensitivity without radioactivity.
- Phospho-specific antibodies allow site-specific analysis.
- Signal to Noise
- Dramatically increased signal to noise ratio over conventional IP/kinase assays.
- Low Background Activity
- Complete System
- Includes everything needed to assay kinase activity.
Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs, such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3), and research investigators consider it an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors, such as U0126 and PD98059.
- Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
- Baccarini, M. (2005) FEBS Lett 579, 3271-7.
- Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
- Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
- Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
- Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
- Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
- Marais, R. et al. (1993) Cell 73, 381-93.
- Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
- Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
- Russell, K.S. et al. (2000) Proc Natl Acad Sci U S A 97, 5930-5. Applications: In vitro kinase assay.
- Tan, J. et al. (2000) J Neurosci 20, 7587-94. Applications: In vitro kinase assay.
- Jost, M. et al. (2001) J Biol Chem 276, 6320-6. Applications: In vitro kinase assay.
- Skapenko, A. et al. (2001) J Immunol 166, 4283-92. Applications: In vitro kinase assay.
- Gui, Y. and Zheng, X.L. (2003) J Biol Chem 278, 53017-25. Applications: In vitro kinase assay.
- Hsu, H.Y. et al. (2004) J Immunol 173, 5989-99. Applications: In vitro kinase assay.
- Wong, C.H. et al. (2005) Endocrinology 146, 1192-204. Applications: In vitro kinase assay.
- He, Z. et al. (2005) J Biol Chem 280, 2446-54. Applications: In vitro kinase assay.
- Schlezinger, J.J. et al. (2006) Toxicol Sci 92, 433-44. Applications: In vitro kinase assay.
- Jia, W. et al. (2006) Mol Cancer Res 4, 549-62. Applications: In vitro kinase assay.
- Zampieri, C.A. et al. (2007) J Virol 81, 1230-40. Applications: In vitro kinase assay.
- Ha, U. et al. (2007) J Immunol 178, 1736-47. Applications: In vitro kinase assay.
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For Research Use Only. Not For Use In Diagnostic Procedures.
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