Preparative isoelectric focusing of proteins using binary buffers in a vortex-stabilized, free-flow apparatus.

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.