Staining acidic phosphoproteins (phosvitin) in electrophoretic gels.

The principal phosphoglycoproteins of avian and amphibian egg yolk, known as phosvitins, have been examined extensively in developmental studies of estrogenic induction of protein synthesis (1) and, more recently, as substrates for protein kinases which phosphorylate the basic nuclear proteins (2,3). Phosvitin has few aromatic and basic amino acids (4) which can be detected by uv absorbance or by conventional anionic protein stains following electrophoresis. In addition, the high negative-charge density of clustered phosphorylserine residues (2) may prevent staining by charge repulsion of anionic dyes. Dilute solutions of cationic dyes like toluidine blue and acridine orange have been employed (5,6), but, in our experience, they penetrate gels slowly and also stain the residual negative charges of most electrophoretic matrices, including agarose, starch, and even polyacrylamide. The only specific method for localizing phosphoproteins in polyacrylamide gels requires several time-consuming steps to liberate orthophosphate by basic hydrolysis, to form an insoluble phosphomolybdate complex, and, finally, to stain this complex with methyl green dye (7). The exceptional affinity of contiguous phosphorylserines for trivalent metal ions (8) can, however, be exploited to detect phosvitin in gels. This strategem may also provide an alternative method for visualizing similar acidic phosphoproteins which have now been identified in brain, spermatozoa, and adrenal medulla (9,lO).