Geahlen R L, Anostario M, Low P S, Harrison M L
Anal Biochem. 1986 Feb 15;153(1):151-8. doi: 10.1016/0003-2697(86)90074-6.
A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.
本文描述了一种在十二烷基硫酸钠-聚丙烯酰胺凝胶上进行电泳后，鉴定蛋白质样品中蛋白激酶活性的方法。通过凝胶内酶的复性，然后用聚丙烯酰胺凝胶中包含的底物或激酶本身与[γ-32P]ATP进行磷酸化反应来检测蛋白激酶活性。接着，在阴离子交换树脂存在下洗涤凝胶以去除未反应的[γ-32P]ATP后，通过放射自显影观察蛋白激酶活性的位置（Mr）。使用纯化的cAMP依赖性蛋白激酶催化亚基进行的研究表明，在含有1mg/ml酪蛋白的凝胶上，低至0.01微克的酶浓度也能轻松检测到。该技术也可用于鉴定多亚基酶的活性亚基。例如，酪蛋白激酶II的活性亚基可以通过在含有酪蛋白的凝胶中使解离的酶复性来轻松确定。蛋白质粗混合物中存在的假定蛋白激酶在通过凝胶电泳分离后也能被检测到，并可根据分子量和磷酸化氨基酸的身份进行表征。使用该技术，在通过红细胞膜亚分级制备的蛋白质混合物中至少检测到了三种主要的蛋白激酶。
