Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double-staining technique.
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 yellow-stained bands without affecting the CB-stained polypeptide pattern. These observations afforded evidence that the yellow-staining bands were sialoglycoproteins and lipids. The double-staining 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.
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004910:Erythrocyte Membrane / Q000032:analysis*; D008563:Membrane Lipids / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D009439:Neuraminidase / Q000378:metabolism; D012394:Rosaniline Dyes; D012795:Sialoglycoproteins / Q000032:analysis*; D012834:Silver; D013194:Staining and Labeling; D014357:Trypsin
D008563:Membrane Lipids; D008565:Membrane Proteins; D012394:Rosaniline Dyes; D012795:Sialoglycoproteins; D012834:Silver; D009439:Neuraminidase; D014357:Trypsin; C004692:coomassie Brilliant Blue