Micropreparative protein purification by reversed-phase high-performance liquid chromatography

Abstract

Micropreparative purification of the four subunits of phosphorylase kinase (molecular weights 16,680, 43,000, 113,000 and 132,000) by reversed-phase high-performance liquid chromatography has provided quantities sufficient for some of the first structural studies of these proteins. The best yield from a 25 × 0.46 cm I.D. column was obtained on a packing material (5μm C 18) bonded under conditions resulting in a relatively low ligand density; of a total 250 μg of protein applied, 76% was recovered. Low recoveries from 5–30-μg sample loads suggest partial irreversible adsorption. Retention of protein by the column after an initial micropreparative separation adversely affects resolution and recovery on subsequent separations. Incorporation of gradient wash steps between sample injections was necessary to maintain column performance and to prolong usable column lifetime. The use of a short column with a large diameter (6 × 1 cm) gave increased loading capacity above 400μg protein and enhanced recovery while maintaining good resolution. A less expensive reversed-phase support (17-μm C 18) provided adequate resolution for this separation.