title: Using genetically engineered kinases to screen for novel protein kinase substrates: phosphorylation of substrates in cell lysates with exogenous kinase.
abstract: INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves the addition of recombinant mutant kinase and [γ-(32)P]ATP analog to cell lysates. This technique has been successfully used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates in cell lysates from SKOV-3 ovarian cells; 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