title: Using genetically engineered kinases to screen for novel protein kinase substrates: phosphorylation of kinase-associated substrates.
abstract: INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-(32)P]ATP analog. This technique has been used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.
authors.forename: Scott T
authors.initials: ST
authors.lastname: Eblen
authors.forename: N Vinay
authors.initials: NV
authors.lastname: Kumar
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Weber