Physicochemical
and structural studies of proteins demand that the protein be of extremely high purity. In addition, rather large quantities of the protein may be required. The apparatus described below offers excellent separation ,of large quantities of closely related proteins.
Ion-exchange column chromatography is probably the most commonly used method of protein purification. Frequently, however, there may be considerable overlap among closely related proteins. This overlap may require the investigator to rerun the pooled fractions on the same column with, perhaps, a different elution scheme (1). Each run on an ion-exchange column may result in an irreversible adsorption of some of the protein on the column, and thereby diminish the yield. The rechromatography may also be very time consuming. Electrofocusing
(2) has recently been used to separate proteins, but the cost of the apparatus and materials may be a deterrent to its frequent use as a preparative method. The time involved in electrofocusing may also be quite extensive. Preparative acrylamide gel electrophoresis (3) has also been used for the purification of proteins, but sample sizes are limited. There is also a tendency for the slower migrating proteins to be greatly diluted and emerge in a very broad peak.
The electrophoresis apparatus described here offers a convenient, inexpensive, and relatively rapid method ,of purifying large quantities of proteins. Virtually 100% of the protein under study can be recovered. This apparatus has been very effective in separating closely related horseradish peroxidase isoenzymes and promises to be of great value in the purification of other proteins.
Methods
The preparative column electrophoresis apparatus is shown schematically in Fig. 1. The major components of the system are two Lucite