Effects of increased voltage on resolution in preparative isoelectric focusing of myoglobin varia.

IEF is a powerful technique which separates proteins and other amphoteric solutes in a pH gradient according to their pI's. The current work evaluates the effect on resolution of increasing electric fields in a novel preparative, vortex-stabilized electrophoresis device. In shallow gradients spanning one pH unit, the variants of myoglobin were separated at applied voltages from 10 to 15 kV. Digital imaging of these separations indicated a 20% reduction in bandwidth and a 60% increase in resolution as the electric field strength is varied across this range. These results were confirmed by IEF-PAGE and ion-exchange chromatography.