Cell Signaling Technology

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 each

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

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 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.

  1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
  2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
  3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
  4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
  5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
  6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
  7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
  8. Marais, R. et al. (1993) Cell 73, 381-93.
  9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
  10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

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

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