Protein Focusing in a Conductivity Gradient

Abstract

Conductivity gradient focusing (CGF) is one member of a family of gradient focusing techniques, characterized by two opposing forces which produce a dynamic equilibrium and which are able to simultaneously separate and concentrate proteins. In CGF, the two counteracting forces result from a constant convective flow of buffer opposed by an electric field gradient. This gradient in the electric field is formed by gradually decreasing buffer conductivity, i.e., when a slow‐moving, relatively high conductivity buffer is dialyzed against a low conductivity purge buffer. This paper presents the design of an analytical‐scale CGF device and the results of several experiments with colored proteins, both in free solution and with the use of a 45 μm size‐exclusion (SEC) packing to decrease dispersion. Experimental results with hemoglobin suggest that CGF may one day be capable of resolving proteins with small charge differences. A linear computer model of conductivity gradient focusing is derived, and some suggestions are made for further development of this new electrophoretic method.