MAP Kinase Signaling Protocols
Springer Science & Business Media, 2004 - Science - 332 pages
Mitogen-activated protein kinase (MAPK) signaling cascades initiate highly significant cellular processes and are involved in a large number of diseases, including cancer and diabetes. In MAP Kinase Signaling Protocols, leading researchers with extensive hands-on experience describe in detail both classic and cutting-edge techniques for the detection and measurement of the MAPKs and other components in these cascades. The protocols include methods for the determination of the subcellular localization of these components, the structural and biophysical analysis of the components, and identification of novel components of known and unknown signaling cascades. Additional methods examine the upstream mechanisms of activation of MAPK cascades by various receptors, the mechanisms involved in the downregulation of the MAPK cascades, and identification of targets of the MAPK cascades. A number of techniques use inhibitors in research on MAPK and transgenic mice for studies of MAPK signaling. Each readily reproducible protocol is described in step-by-step detail and includes a background introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and versatile, MAP Kinase Signaling Protocols offers biochemists, cell biologists, physicians, and biotechnologists all the essential laboratory techniques needed to conduct productive studies of MAPK signaling in health and disease, in measuring the influence of drugs, and in a broad range of experimental systems.
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agarose amino acids antibody aprotinin beads binding Biol BMK1 c-Jun cDNA cell lysate cellular centrifuge Chem clones column containing detection diluted domain electrophoresis enzyme ERK cascade ERK1 ERK2 expression extracellular extracellular signal-regulated kinase extract filters gene growth factor GST-PTP-SL ice-cold immunoblot immunoprecipitation Incubate inhibitors interaction IPTG isoforms kinase activity kinase assay kinase kinase ligand lysis buffer mammalian MAP Kinase Signaling MAP3K MAPK activation MAPK cascades medium MEK1 MEKK1 MEKs membrane method mg/mL mitogen-activated protein kinase molecular motif mutant N-terminal NaCl nitrocellulose nuclear oocytes p38MAPK peptide library phage phosphatases phosphorylation plasmid plates PMSF prepared PTP-SL purified reaction reagents receptor recombinant regulation residues room temperature SAPK/JNK screening SDS-PAGE Seger sequence serum Sigma signaling pathways solution specific stimulation Subheading substrate supernatant tein tion transcription factor transfection transfer Tris-HCl Triton tube tyrosine ubiquitination vector vitro washing buffer Western blot