Measuring MAP kinase activity in immune complex assays.
I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins.
Cherkasova
Vera A
VA
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. cherkasv@mail.nih.gov
D015153:Blotting, Western / Q000379:methods; D002457:Cell Extracts; D007118:Immunoassay / Q000379:methods*; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism
D002457:Cell Extracts; D029701:Saccharomyces cerevisiae Proteins; C075580:FUS3 protein, S cerevisiae; C088589:HOG1 protein, S cerevisiae; C541984:KSS1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae