Cell Signaling Technology

Product Pathways - MAPK Signaling

p38 MAP Kinase Assay Kit (Nonradioactive) #9820

Kit Includes Quantity Source
Immobilized Phospho-p38 MAPK (Thr180/Tyr182) Mouse mAb # 9219 800 microliters Mouse
ATF-2 Fusion Protein # 9224 80 micrograms Rabbit
Kinase Buffer (10X) # 9802 15 milliliters
Cell Lysis Buffer (10X) # 9803 15 milliliters
ATP (10 mM) # 9804 50 microliters
Anti-rabbit IgG, HRP-linked Antibody # 7074 50 microliters Goat
Anti-biotin, HRP-linked Antibody # 7075 100 microliters Goat
Biotinylated Protein Ladder Detection Pack # 7727 100 microliters
20X LumiGLO® Reagent and 20X Peroxide # 7003 5 milliliters
20X LumiGLO® Reagent and 20X Peroxide # 7003 5 milliliters
Phospho-ATF-2 Ab 100 microliters

Molecular Weight

Expected Molecular Weight: 43kDa

Reactivity

H M

Reactivity Key:  H=Human  M=Mouse

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 anisomycin-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 2 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 which participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAP kinase, 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 MAP kinase is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), UV light and growth factors (1-5). MKK3, MKK6 and SEK activate p38 MAP kinase by phosphorylation at Thr180 and Tyr182. Activated p38 MAP kinase 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).

  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.

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