CHK1 kinase activity assay.
CHK1 kinase activity assay.
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CHK1激酶活性测定。
DOI：
10.1007/978-1-61779-998-3_39
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Wang,Ya
中科院分区：
文献类型：
--
作者：
Wang,HongYan;Wang,Ya
哺乳动物CHK 1是一种Ser/Thr激酶，在ATR（ATM和Rad 3相关蛋白激酶）下游的DNA损伤激活的细胞周期检查点信号通路中起关键作用。本章重点描述了一种测定CHK 1体外活性的方法。本试验的基本机制是观察含有可被CHK 1体外磷酸化位点的CDC 25 C片段的磷酸化水平。该测定包括五个主要步骤：（1）从对照或处理的细胞制备提取物，（2）制备底物，（3）从细胞中免疫沉淀CHK 1蛋白，（4）组装激酶测定，（5）通过CHK 1分析底物的磷酸化水平。除了CHK 1，CHK 2是另一个重要的检查点调节因子，对DNA损伤做出反应。由于CHK 1和CHK 2在体外共享一些底物（如CDC 25 C），因此该测定也可用于CHK 2活性测定，但CHK 2抗体将被CHK 1抗体替代。
Mammalian CHK1 is a Ser/Thr kinase that plays a critical role in the DNA damage-activated cell cycle checkpoint signaling pathway downstream of ATR (ATM and Rad3 related protein kinase). This chapter focuses on describing an assay to measure CHK1 activity in vitro. The basic mechanism of this assay is to observe the phosphorylated levels of a fragment of CDC25C containing the site that can be phosphorylated by CHK1 in vitro. This assay includes five major steps: (1) preparing extracts from the control or treated cells, (2) preparing substrate, (3) immunoprecipitating CHK1 protein from the cells, (4) assembling the kinase assay, (5) analyzing the phosphorylated level of the substrates by CHK1. Besides CHK1, CHK2 is another important checkpoint regulator that responds to DNA damage. Because CHK1 and CHK2 share some substrates such as CDC25C in vitro, this assay could also be used for a CHK2 activity assay, except that the CHK2 antibody will be replaced by the CHK1 antibody.