A new technique for eluting proteins from polyacrylamide gels: Flat‐bed electrophoretic elution in Sephadex via isoelectric focusing

Abstract

AbstractWe have developed a new technique, flat‐bed electrophoretic elution in Sephadex via isolectric focusing for the removal of proteins from polyacrylamide gels subsequent to electrophoresis. A narrow slice of the polyacrylamide gel, which contains the protein to be eluted, is finely ground and applied to a horizonatal bed of Sephadex G‐200 SF containing carrier ampholytes. Upon focusing the Sephadex plate, the protein rapidly elutes and migrates to its isoelectric point where it bands and concentrates. This method, by circumventing many of the problems and limitations of current elution methods and apparatus, ensures the recovery of proteins in concentrated form with a high yield.Conditions for optimal rates of elution were developed. For best results, a finely chopped slice of the gel, which contains the protein, is mixed in with some of the Sephadex G‐200 SF which has been removed from a narrow rectangular application zone of a flat‐bed plate. The application zone is chosen several pH units from the isoelectric point of the eluting material. The use of a broad pH carrier ampholyte in the bed generally proves superior to a narrow range interval. High voltage can be used due to the excellent cooling capacity of the system and the even conductivity of the Pharmalyte® carrier ampholytes. The technique is relatively mild and should only minimally influence the biological activity of the eluted material.