In-gel kinase assay as a method to identify kinase substrates.
Phosphorylation of proteins by kinases is central to many cellular processes, including signal transduction. Thus, assays to identify or characterize kinases are a key tool for research in this area. Kinase substrates can be incorporated into polyacrylamide gels and used to characterize kinase activity in mixed samples. This methodology can be adapted for the identification of novel kinase-substrates or kinase-kinases that participate in the regulation of cell signaling. Here, I review the rationale and principles of an in-gel kinase assay. This strategy relies on co-polymerization of a substrate within the gel matrix, followed by detection of enzymatic activity in situ. The following Protocol provides a detailed method for performing the in-gel kinase assay and discusses the uses of the assay to evaluate kinase activity in the context of proliferation, differentiation, and survival pathways.
Wooten
Marie W
MW
Department of Biological Sciences, Program in Cell Molecular Biosciences, Auburn University, Auburn, AL 36849, USA. wootemw@auburn.edu
D001345:Autoradiography / Q000379:methods; D002021:Buffers; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002457:Cell Extracts / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D020932:Nerve Growth Factor / Q000494:pharmacology; D010766:Phosphorylation; D011108:Polymers / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D011489:Protein Denaturation; D011494:Protein Kinases / Q000378:metabolism*; D020673:Protein Renaturation; D011506:Proteins / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013379:Substrate Specificity
D002021:Buffers; D002457:Cell Extracts; D011108:Polymers; D011480:Protease Inhibitors; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D020932:Nerve Growth Factor; D011494:Protein Kinases