Recent developments in preparative free flow isoelectric focusing

Continuous flow electrophoresis (CFE) is a promising method for preparative fractionation of a variety of biological species, ranging from peptides and proteins to subcellular particles and cells. The high separation efficiency of FFE may be deteriorated by hydrodynamic distortion of zones due to the omnipresent parabolic laminar flow profile. We show in this paper that the detrimental hydrodynamic distortion of separated proteins zones can be reduced, with resultant enhancement of separation efficiency, by employing continuous isoelectric focusing in pH gradients as the actual working regime in an advanced instrumentation. Newly developed media for fast generation of narrow- or broad-range pH gradients under CFE conditions are described. The separation efficiency of these pH gradients is comparable to that of the gradients formed with the aid of synthetic carrier ampholytes. The new media are defined mixtures of nontoxic chemicals, and thus they are compatible with the requirements of human medicine. Experimental data are given showing that the new media offer fractionation of isoforms of proteins, that they offer resolution of proteins differing in isoelectric point (pI) by less than 0.05 pH units, and that these media inhibit proteins precipitation in experiments with human serum proteins.