Product Pathways - MAPK Signaling
p38 MAP Kinase Assay Kit (Nonradioactive) #9820
|9820||carrier free and custom formulation / quantity||email request|
|Human, Mouse, Rat|
|Phospho-p38 MAPK (Thr180/Tyr182) (28B10) 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|
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.
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.
- 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.
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).
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- Lee, J.C. et al. (1994) Nature 372, 739-746.
- Freshney, N.W. et al. (1994) Cell 78, 1039-1049.
- Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.
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- Zhao, M. et al. (1999) Mol. Cell. Biol. 19, 21-30.
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- Cuenda, A. et al. (1995) FEBS Lett 364, 229-33.
- Kumar, S. et al. (1999) Biochem Biophys Res Commun 263, 825-31.
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