Two-Phase Electrophoresis of Proteins

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Abstract
Proteins have been directed into either the top or bottom phase of a polyethylene glycol/dextran aqueous two-phase system by applying an electric field perpendicular to the phase interface. Protein migration across the interface was manipulated by varying polarity, pH, electrophoresis time, field strength, and phase volume ratio. Mixtures of hemoglobin and albumin were separated by operating between isoelectric points and directing oppositely charged proteins into separate phases. Applying 50 V/cm for 2 hours to 58 mL of an equal phase volume two-phase system containing 0.2 g/L of each protein at pH 6 resulted in a bottom phase containing 99% of the hemoglobin and a top phase containing 95% of the albumin. This represents a significant improvement over the separation obtained either by partitioning in the same two-phase system with no applied field or by electrophoresis under the same conditions in homogeneous buffer. The two-phase system divides the electrophoresis device into two distinct regions, providing stability against convective mixing and facilitating product isolation.