Please cite as: CSH Protocols; 2007; doi:10.1101/pdb.prot4639

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Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Substrates in Cell Lysates with Exogenous Kinase

Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber

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 [{gamma}-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.


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Related Protocols

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Generation of [{gamma}-32P]ATP Analog from ADP Analog
Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber
CSH Protocols 2007: 4637. [Abstract] [Full Text]

Using Genetically Engineered Kinases to Screen for Novel Protein Kinase Substrates: Phosphorylation of Kinase-Associated Substrates
Scott T. Eblen, N. Vinay Kumar, and Michael J. Weber
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