Separation of serum proteins by using a simple thin layer polyacrylamide gel electrophoresis

Abstract

Although the thin layer polyacrylamide gel electrophoresis is a highly suitable and satisfactory method for the analysis of serum proteins, the procedure is not so simple as that of cellulose acetate membrane electrophoresis.The authors have been studying on the micro-electrophoresis of proteins and enzymes in serum and have accomplished a procedure for “Simple polyacrylamide gel film electrophoresis” of high resolution. The procedure and some of the results were reported.It is based on thin layer electrophoresis on a sheet coated by the thin layer of polyacrylamide gel (1mm or 2mm thick), which has been previously prepared and kept in moist condition. Therefore, preparation of thin layer could be omitted from the procedure.As the sheets, cellophane of a quality comparable to dialysis tubing, 124 PD cellophane (Du Pont Co.), was selected. A polyester film used for drawing with a smooth surface and a rough back was also found suitable in regard to the firm adhesion of the gel layer to the sheet, since the gel layer could be formed on the rough side.Various buffer conditions of gel layer could be obtained by using monomer buffer mixture solution which was prepared by dissolving 9.5g of acrylamide and 0.5g of BIS (N, N'-methylenebisacrylamide) in 200ml of 10% glycerol buffer solution. The gelation was performed by adding catalysts to monomer buffer mixture solution.Sera were applied without dilution into the sample grooves previously prepared on the gel layer surface. 3μl or 10-15μl of serum was sufficient for each groove on the gel layer with 1mm or 2mm thickness, respectively.The gel layer on a supporting glass plate bridged between the two electrode compartments and the ends of the gel were connected by wet filter papers to the buffer solution in the compartments.To prevent over-heating, the electrophoresis was carried out at 5°C to 10°C. The voltage was adjusted to deliver a constant current of 0.5 to 1.0mA per cm width of the gel layer and the current was applied for 120min.Various staining methods for enzyme and protein components could be applied after electrophoresis.The stained gel layer was inserted between the sheets of cellophane, and the gel layer was plastized by leaving at room temperature for two days.The procedure was also found to be a useful tool for the estimation of molecular weights of polypeptide chains by using the gel layer containing sodium dodecyl sulfate (SDS).