Hastie C James, McLauchlan Hilary J, Cohen Philip
Division of Signal Transduction Therapy, Medical Sciences Institute-Wellcome Trust Biocentre Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland.
Nat Protoc. 2006;1(2):968-71. doi: 10.1038/nprot.2006.149.
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.
蛋白激酶活性导致放射性标记的磷酸基团从[γ-32P]ATP掺入到肽或蛋白质底物中。测量作为时间和酶浓度函数掺入底物中的放射性量可对酶活性进行定量。活性以“单位”表示，其中1个单位对应于在1分钟内催化1纳摩尔磷酸掺入标准底物的蛋白激酶量。比活性定义为每毫克蛋白质的活性单位。此处描述的测定形式快速、简单、廉价、灵敏且准确，可直接测量活性，并且仍然是蛋白激酶活性定量的“金标准”。一次最多可手动检测40个样品，一个人完成该检测所需时间不到1小时。
