Assay of protein kinases using radiolabeled ATP: a protocol
Assay of protein kinases using radiolabeled ATP: a protocol
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DOI：
10.1038/nprot.2006.149
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Cohen, Philip
中科院分区：
文献类型：
--
作者：
Hastie, C. James;McLauchlan, Hilary J.;Cohen, Philip
蛋白激酶活性导致[γ-P-32] ATP的放射性标记磷酸盐掺入肽或蛋白质底物中。作为时间和酶浓度的函数的掺入底物中的放射性的量的测量允许定量酶活性。活性表示为“单位”，其中1单位对应于催化1纳摩尔磷酸盐在1分钟内掺入标准底物的蛋白激酶的量。比活性定义为每毫克蛋白质的活性单位。本文描述的测定形式快速、简单、廉价、灵敏和准确，提供了活性的直接测量，并且仍然是蛋白激酶活性定量的“金标准”。一次可手动检测多达40个样品，一个人不到1小时即可完成检测。
Protein kinase activity results in the incorporation of radiolabeled phosphate from [gamma-P-32] 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.