Enhancement of the detection of alkali-resistant phosphoproteins in polyacrylamide gels

Following their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, labeled proteins obtained from cultured canine prostatic epithelial cells incubated with [ 35S]-methionine and [ 32P]phosphate were subjected to alkali treatment, a method that is currently used to detect phosphotyrosine-containing proteins. Significant amounts of 35S-labeled material were lost during the alkali treatment. The crosslinking of proteins within the gels by glutaraldehyde treatment eliminated protein losses and did not alter the efficiency of phosphoester bond hydrolysis by alkali treatment. Consequently, the time required to detect alkali-resistant phosphoproteins by autoradiography was greatly reduced. Prostatic phosphoproteins were also shown to contain phosphotyrosine, indicating the presence of tyrosine protein kinase activity in these proliferating epithelial cells.