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
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).
- Rouse, J. et al. (1994) Cell 78, 1027-1037.
- Han, J. et al. (1994) Science 265, 808-811.
- 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.
- Zervos, A.S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10531-10534.
- Zhao, M. et al. (1999) Mol. Cell. Biol. 19, 21-30.
- Yang, S.H. et al. (1999) Mol. Cell. Biol. 19, 4028-4038.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
