Product Pathways - MAPK Signaling
p44/42 MAP Kinase Assay Kit (Nonradioactive) #9800
| Kit Includes | Quantity | Source |
|---|---|---|
| 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 |
Molecular Weight
41, 45 kDa
Species Cross-Reactivity
H M R
Reactivity Key:
H=Human
M=Mouse
R=Rat
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.
Description
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.
Western Blotting
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.
- Specificity
- 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.
Background
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 is 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.
Application References
- 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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Protocols
- 3510:
- Immunoprecipitation
- 9186:
- Western Blotting
Companion Products
- 9101 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody
- 9211 Phospho-p38 MAPK (Thr180/Tyr182) Antibody
- 9221 Phospho-ATF-2 (Thr71) Antibody
- 9231 Phospho-MKK3 (Ser189)/MKK6 (Ser207) Antibody
- 9151 Phospho-SEK1/MKK4 (Thr261) Antibody
- 7720 Prestained Protein Marker, Broad Range (Premixed Format)
- 7727 Biotinylated Protein Ladder Detection Pack
This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.