Assay of protein kinases using radiolabeled ATP: a protocol.
Protein kinase activity results in the incorporation of radiolabeled phosphate from [gamma-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 quantified. 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. Specific activity is defined 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 quantification 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.
Hastie
C James
CJ
Division of Signal Transduction Therapy, Medical Sciences Institute-Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
McLauchlan
Hilary J
HJ
Cohen
Philip
P
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D007553:Isotope Labeling; D010758:Phosphorus / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D011494:Protein Kinases / Q000378:metabolism*; D012680:Sensitivity and Specificity
D010761:Phosphorus Radioisotopes; D010758:Phosphorus; D000255:Adenosine Triphosphate; D011494:Protein Kinases