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Assay of protein kinases using radiolabeled ATP: a protocol
C James Hastie1,3, Hilary J McLauchlan1,3 & Philip Cohen2
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1Division of Signal Transduction Therapy and 2Medical Research Council Protein Phosphorylation Unit, Medical Sciences InstituteWellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland. 3These authors contributed equally to this manuscript. Correspondence should be addressed toP.C. ([email protected]).
Published online 3 August 2006; doi:10.1038/nprot.2006.149
Protein kinase activity results in the incorporation of radiolabeled phosphate from [c-32P]ATP into a peptide or protein substrate. The measurement of the amount of radioactivity incorporated into a substrate as a function of time and enzyme concentration allows enzyme activity to be quantied. The activity is expressed as a unit, where 1 unit corresponds to the amount of protein kinase that catalyzes the incorporation of 1 nanomole of phosphate into the standard substrate in 1 minute. Specic activity is dened as units of activity per milligram protein. The assay format described here is quick, simple, inexpensive, sensitive and accurate, provides a direct measurement of activity and remains the gold standard for the quantication of protein kinase activity. Up to 40 samples can be assayed manually at one time, and the assay takes one person less than 1 hour to complete.
INTRODUCTIONThe attachment and removal of phosphate from proteins, called reversible phosphorylation, is catalyzed by two classes of enzyme, called protein kinases and protein phosphatases. Protein phosphorylation regulates nearly all aspects of cell life, and protein kinases are the largest single family of enzymes encoded by the human genome, with over 500 members. Abnormal protein phosphorylation is a cause or a consequence of major diseases worldwide, including cancer, diabetes and chronic inammatory diseases. For that reason, protein kinases have become the second most highly studied class of drug target, behind G proteincoupled receptors (GPCRs), and are a chief area of research for both academic laboratories and the pharmaceutical industry1. Protein kinases catalyze the phosphorylation of serine, threonine, tyrosine and histidine residues on their target proteins, a reaction that involves the transfer of the g-phosphoryl group of ATP to the amino...