An updated two-dimensional gel electrophoresis technique for the detectionof drug-induced changes in protein phosphorylation in intact smooth muscle

Two-dimensional gel electrophoresis is widely used in many areas of scienti fic research. The necessity for greater resolution and more sensitive prote in detection with this technique have resulted in a steadily changing metho dology. Complete descriptions of some aspects of two-dimensional gel electr ophoresis are available in the earlier literature. However, simplified meth ods incorporating recent advances specifically designed to use two-dimensio nal gel electrophoresis for the measurement of protein phosphorylation in i ntact tissue are lacking. This report describes, in detail, each of the ste ps involved in carrying out such measurements including intact tissue label ing with P-32, homogenization and protein sample preparation, two-dimension al gel electrophoresis using isoelectric focusing followed by vertical seco nd-dimension SDS-PAGE, staining, autoradiography, and quantitative analysis of changes in phosphorylation of specific proteins. This method incorporat es a number of modifications taken from other published sources and include s several novel changes developed in our laboratory. To illustrate the util ity of this technique we have included a set of results analyzing the phosp horylation patterns induced by the addition of a nitric oxide donor, sodium nitroprusside, to intact strips of rat aorta. We were able to demonstrate SNP-induced phosphorylation of a number of proteins, several of which have nut been previously described in earlier reports in which the patterns of P KG-mediated phosphorylation were investigated. (C) 2000 Elsevier Science In c. All rights reserved.