Rapid purification of clathrin-coated vesicles by free-flow electrophoresis

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.