Eblen Scott T, Kumar N Vinay, Weber Michael J
CSH Protoc. 2007 Apr 1;2007:pdb.prot4638. doi: 10.1101/pdb.prot4638.
INTRODUCTIONThis protocol describes a method for detection of direct substrates of a protein kinase in cell lysates or fractions. The approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [γ-(32)P]ATP analog. This technique has been used for the phosphorylation of extracellular signal-regulated kinase 2 (ERK2) substrates; however, the methodology can be applied to other protein kinases as well.
引言
本方案描述了一种在细胞裂解物或组分中检测蛋白激酶直接底物的方法。该方法包括通过从转染的COS-1细胞中免疫沉淀突变激酶的标记形式来鉴定激酶相关底物，并通过添加[γ-(32)P]ATP类似物进行激酶反应。该技术已用于细胞外信号调节激酶2（ERK2）底物的磷酸化；然而，该方法也可应用于其他蛋白激酶。
