Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Substrates in Cell Lysates with Exogenous Kinase
This protocol was adapted from “Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates,” Chapter 24, in Protein-Protein Interactions (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
This 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-32P]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.
