Isoelectric focusing of proteins in capillary electrophoresis with pressure-driven mobilization

Abstract

An isoelectric focusing (IEF) method in the capillary format with wide linear pH range (pH 3–10) and high resolution has been developed for separations of proteins. The methodology involves applying pressure and voltage simultaneously during the mobilization step of the IEF process, to elute the focused protein zones for detection. Capillary isoelectric focusing (CIEF) is performed in neutral, hydrophilic, coated capillaries of 50 μm I.D., using various concentrations of methyl cellulose to reduce the distortion of focused zone by parabolic laminar flow. A linear relationship between the retention time and isoelectric point (pI) of protein standards was observed. The high resolution of this technique was demonstrated by the separation of hemoglobin variants F and A with pI difference of 0.05 pH units, and by the resolution of the isoforms of an anti-carcinoembryonic antigen monoclonal antibody.