Upside-Down Stopped-Flow Electrofractionation of Complex Protein Mixtures

Abstract

The excellent resolution of SDS–PAGE in protein analysis stimulated the creation of various preparative devices. The main approach used in these devices is the construction of a elution chamber in the lower end of the polyacrylamide gel cylinder or plate. Although this continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis, some limitations to this approach exist. There is strong dilution of protein zones by the eluting buffer, which drastically restricts the sensitivity of the determination of minor proteins, and the restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. To overcome these problems, the upside-down stopped-flow electrofractionation system (UDSFE) was designed. The necessary quantity of fraction is drawn with a pipet in a small volume from just above the gel cylinder. This invention improves the possibility of electrofractionation of deluted complex protein mixtures. The efficiency of this technique is demonstrated by purification a protein kinase from rat liver. The method has also been successfully used for purification of error-correcting 3′–5′ exonuclease.