An updated two-dimensional gel electrophoresis technique for the detection of drug-induced changes in protein phosphorylation in intact smooth muscle.
Two-dimensional gel electrophoresis is widely used in many areas of scientific research. The necessity for greater resolution and more sensitive protein detection with this technique have resulted in a steadily changing methodology. Complete descriptions of some aspects of two-dimensional gel electrophoresis are available in the earlier literature. However, simplified methods incorporating recent advances specifically designed to use two-dimensional gel electrophoresis for the measurement of protein phosphorylation in intact tissue are lacking. This report describes, in detail, each of the steps involved in carrying out such measurements including intact tissue labeling with 32P, homogenization and protein sample preparation, two-dimensional gel electrophoresis using isoelectric focusing followed by vertical second-dimension SDS-PAGE, staining, autoradiography, and quantitative analysis of changes in phosphorylation of specific proteins. This method incorporates a number of modifications taken from other published sources and includes several novel changes developed in our laboratory. To illustrate the utility of this technique we have included a set of results analyzing the phosphorylation 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 not been previously described in earlier reports in which the patterns of PKG-mediated phosphorylation were investigated.
Hennan
J K
JK
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, British Columbia, Canada.
Diamond
J
J
D000818:Animals; D017869:Cyclic GMP-Dependent Protein Kinases; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D007525:Isoelectric Focusing; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D011506:Proteins; D009599:Nitroprusside; D011494:Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases