Valtorta F, Schiebler W, Jahn R, Ceccarelli B, Greengard P
Anal Biochem. 1986 Oct;158(1):130-7. doi: 10.1016/0003-2697(86)90600-7.
A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides.
本文描述了一种用于磷蛋白磷酸化和检测的新方法。从组织样品中溶解蛋白质，通过聚丙烯酰胺凝胶电泳进行分离，转移到硝酸纤维素膜滤器上，然后用环磷酸腺苷（腺苷3':5'-单磷酸）依赖性蛋白激酶的催化亚基对印迹的多肽进行磷酸化。该方法是为检测去磷酸化突触素I而开发的，但也已证明适用于其他蛋白质的磷酸化。使用新方法磷酸化的组织样品的磷酸化模式与使用传统试管检测获得的模式相似。一旦磷酸化，吸附的蛋白质可以用蛋白酶消化并进行磷酸肽图谱分析。磷酸化的印迹蛋白质也可以通过覆盖技术进行分析，以进行多肽的免疫检测。
