GB1060874A - Method and apparatus for use in the detection by staining of substances separated by zone electrophoresis - Google Patents

Abstract

<PICT:1060874/C6-C7/1> A method of detecting components of a mixture in colloidal solution, e.g. in the analysis of biological fluids such as blood serum, comprises the steps of separating the components by zone electrophoresis in an anti-convective supporting electrophoretic medium, treating the supporting medium with a dye which preferentially stains the separated components, followed by the step of carrying out a further electrophoretic treatment by an electric field applied in a direction substantially perpendicular to the direction of initial electrophoretic separation and in the presence of a dye-adsorbing material, e.g. charcoal, external to the supporting medium so that the surplus dye is caused to migrate to the dye-adsorbing material and is thereby removed from the supporting medium. The supporting electrophoretic medium may consist in a flat rectangular block or slab or a cylindrical body of a gel composed of starch, or a polyacrylamide or other synthetic polymer material. Fig. 1 illustrates electrophoretic removal of surplus dye from gel 22 placed on a powdered charcoal dye-adsorbing bed 5 and between horizontal rod anodes 10 and cathodes 17 e.g. of carbon. A perforated or slotted shelf 2 covered with filter paper 4 supports the charcoal 5 which in turn is covered with filter paper 6 upon which is laid a screen or mask 7 of insulating synthetic plastics material, e.g. polymethylmethacrylate, which surrounds the gel 22. Polymethylmethacrylate may also be used for other structural parts of the apparatus. Fig. 2 (not shown) illustrates the mounting and electrical connection to the rod anodes 10. Fig. 3 (not shown) illustrates the mounting of the rod cathodes 17 in carrier plates 18 joined by spacing rods 19. Alternatively, a cathode may be carried by a lid. Vents 25 at the corners of the cell allow gaseous electrolysis products to escape from beneath the charcoal. The electrolyte used in the apparatus may be a mixture of CH3OH, H2O and glacial CH3COOH, should be a solvent for the staining dye and may provide a pH value at which the protein components will be precipitated within the gel. Currents of 1 amp and 1.5 amps for every 100 cm2 of gel surface for starch and polyacrylamide gels respectively are specified for the apparatus. Gels prepared in a buffer containing urea need adequate soaking in staining solution to assist in reducing the urea content.