Cell Signaling Technology

Product Pathways - MAPK Signaling

p38 MAP Kinase Assay Kit (Nonradioactive) #9820

Kit Includes Quantity Source
Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb (Sepharose Bead Conjugate) #9219 400 µl Mouse
ATF-2 Fusion Protein #9224 40 µg Rabbit
Phospho-ATF-2 (Thr71) Antibody #9221 100 µl Rabbit
Kinase Buffer (10X) #9802 15 ml
Cell Lysis Buffer (10X) #9803 15 ml
ATP (10 mM) #9804 50 µl
Anti-rabbit IgG, HRP-linked Antibody #7074 100 µl Goat
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
20X LumiGLO® Reagent and 20X Peroxide #7003 5 ml each

Molecular Weight

34 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 p38 MAP Kinase Assay Kit provides all the reagents necessary to measure p38 MAP kinase activity in cells. First, Immobilized Phospho-p38 MAPK (Thr180/Tyr182) mAb is used to immunoprecipitate p38 MAP kinase, then an in vitro kinase assay is performed using ATF-2 as a substrate. ATF-2 phosphorylation is detected by Western blotting using Phospho-ATF-2 (Thr71) Antibody.

Western Blotting

Western Blotting

Analysis of p38 MAP Kinase activity of UV-treated NIH-3T3 cells by western blot using Phospho-ATF-2 (Thr71) Antibody. Cell extracts were incubated overnight with Immobilized p38 MAPK (Thr180/Tyr182) mAb. Kinase reaction was performed in the presence of 100 µM of cold ATP and 1 µg of ATF-2 fusion protein. Phosphorylation of ATF-2 at Thr71 was measured by western blot using Phospho-ATF-2 (Thr71) Antibody.

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

p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAPK, p38α, β, γ (also known as Erk6 or SAPK3), and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors (1-5). MKK3, MKK6, and SEK activate p38 MAPK by phosphorylation at Thr180 and Tyr182. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6), and MEF2 (5-8).SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) is a selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10).

  1. Rouse, J. et al. (1994) Cell 78, 1027-1037.
  2. Han, J. et al. (1994) Science 265, 808-811.
  3. Lee, J.C. et al. (1994) Nature 372, 739-746.
  4. Freshney, N.W. et al. (1994) Cell 78, 1039-1049.
  5. Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.
  6. Zervos, A.S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10531-10534.
  7. Zhao, M. et al. (1999) Mol. Cell. Biol. 19, 21-30.
  8. Yang, S.H. et al. (1999) Mol. Cell. Biol. 19, 4028-4038.
  9. Cuenda, A. et al. (1995) FEBS Lett 364, 229-33.
  10. Kumar, S. et al. (1999) Biochem Biophys Res Commun 263, 825-31.

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

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