A silver/Coomassie brilliant blue R-250 staining technique that permits a color-coded differentiation of erythrocyte membrane proteins, sialoglycoproteins, and lipids in a single one-dimensional NaDodSO4/polyacrylamide gel has been described. Gels stained first with silver stain and then with Coomassie blue (CB) showed the characteristic blue staining of all conventional CB-sensitive membrane polypeptides, whereas periodic acid-Schiff reagent-sensitive sialoglycoproteins and lipids stained yellow. Several yellow Ag-stained bands corresponding to major and minor sialoglycoproteins were detected at Mr x 10-3 of 88, 72, 65, 41, 35, 31, 28, 24, and 20. Neuraminidase treatment of intact erythrocytes caused shifts in the electrophoretic mobilities of several yellowstained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellowstaining bands were sialoglycoproteins and lipids. The doublestaining technique was used in a topological analysis of the membrane surface of the erythrocyte using protease digestion and selective solubilization. Trypsin cleaved the yellow bands at Mr 88,000 and 41,000. Membrane-associated cleavage products were noted at Mr 58,000 and 38,000. Pronase treatment of intact cells gave membrane-associated cleavage products at Mr 38,000 (yellow) and two CB-stained bands at Mr 58,000 and 60,000. These results suggested that the double-staining technique may be applicable in compositional and topological analyses of other biological membranes.The successful application of NaDodSO4/polyacrylamide gel electrophoresis as a technique for resolving complex mixtures of macromolecules has particularly enhanced the compositional and topological analysis of biological membranes. One consistent problem encountered in these studies, however, is the nonavailability of simple and sensitive methods for selective detection of the different classes of membrane constituents present in the same gel. Coomassie brilliant blue R-250 (CB) and periodic acid-Schiff (PAS) reagent staining of proteins and sialoglycoproteins (1), respectively, permit visualization of only one major class of membrane constituents in parallel gels. In addition, important compositional and topological information on trace membrane components are often lost because of the low sensitivity of these stains. Alternatively, covalent radioactive ligand labeling of membrane constituents has been used extensively to augment the sensitivity of macromolecule detection in gels (2-5).We now report a silver/CB double-staining technique that permits a simple color-coded detection and direct visualization of human erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same gel without recourse to radioisotopes. The technique to be described uses silver staining of gels first and then CB staining. It differs significantly fromThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with ...
