MAP Kinase, p38 Assay Kit, Nonradioactive, (MAPK) BioAssay(TM)

Kit includes all reagents required to measure p38 MAP Kinase activity, including antibodies, substrate, buffers, and a chemiluminescent detection system. The assay system uses two different phospho- antibodies, one to selectively precipitate active p38 MAP Kinase and a second to detect p38- induced phosphorylation of ATF-2 at Thr71. Signal to noise and overall sensitivity and specificity are improved over conventional IP-kinase assays, and best of all, radioactivity is totally eliminated. A monoclonal phospho- specific antibody to p38 MAP Kinase (Thr180/Tyr182) is used to selectively immunoprecipitate active p38 MAP Kinase from cell lysates. The resulting immunoprecipitate is then incubated with ATF-2 fusion protein in the presence of ATP and Kinase Buffer; this allows immunoprecipitated active p38 MAP Kinase to phosphorylate ATF-2. \n\nPhosphorylation of ATF-2 at Thr71 is measured by Western blotting using a phospho- ATF-2 (Thr71) antibody. Thr71 of ATF-2 is a major ATF-2 phosphorylation site required for transcriptional activity (1). Selective analysis of ATF-2 phosphorylation at Thr71 results in improved specificity with high sensitivity and near zero background. Unlike 32P assays where the sensitivity is limited by the highly diluted 32P label (usually to 50uM ATP), this assay can, in principle, detect every phosphorylated ATF-2 molecule. As a result, these changes dramatically improve signal to noise over conventional 32P assays and reduce autoradiography time to minutes instead of days.\n\nKit Components:\n

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SPECIFICATIONS

Catalog Number

M2352-08

Size

1Kit

Applications

IP

References

US Biological application reference: Wang, S. et al., (2006) Blood 107:2432-2439. 1. Han, J., et al., Science 265: 808-811 (1994). 2. Lee, J.C., et al., Nature 372: 739-746 (1994). 3. Rouse, J., et al., Cell 78: 1027-1037 (1994). 4. Freshney, N.W., et al., Cell 78: 1039-1049 (1994). 5. Raingeuad, J., et al., J. Biol. Chem. 270: 7420-7426 (1995). 6. Zervos, A.S., et al., Proc. Natl. Acad. Sci. USA 92: 10,531-10,534 (1995). 7. Zhao, M., et al., Mol. Cell. Biol. 19: 21-30 (1999). 8. Yang, S.H., et al., Mol. Cell. Biol. 19: 4028-4038 (1999). 8. Banerjee, S., et al., Journal of Virology 76: 5937-5948 (2002).