Separation of normal human erythrocyte membrane proteins by high resolution two-dimensional gel electrophoresis

Different factors influencing two-dimensional gel electrophoresis of red cell membrane proteins were studied: membrane preparation and sample solubilization with a special regard to proteolytic artifacts, urea addition, slab gel acrylamide concentrations and silver staining methods. Spot patterns were analyzed both visually and by means of computer-assisted densitometry. A resolution of around 450 spots was achieved on 10% acrylamide slab gels. The reproducibility of the whole two-dimensional gel electrophoresis procedure was assessed by analysis of computer generated spot densities on gels which were run simultaneously with the same sample. It was shown that the standard red cell membrane preparation method does not lead to proteolysis, contamination by cytosolic proteins, or proteins from other cell types. In comparison with previous studies the relatively high resolution seemed to be due to a high solubilization efficiency combined with the use of a sensitive silver staining method.