6 - Preparative Isoelectric Focusing

Abstract

The net charge of an amphoteric molecule is determined by the pH of its local environment. In the case of protein molecules this is the sum of all positive and negative charges of the ionizable amino acid side chains and prosthetic groups. For every protein there is a specific pH at which the net charge is zero. This isoelectric pH value (pI) is a characteristic physicochemical property of every protein. The amphoteric nature of proteins can be used as the basis for isolectric focusing (lEF). In this method proteins are separated according to their pI in a pH gradient gel. Stable, linear pH gradients are the keys to successful lEF. lEF is a high-resolution technique that can resolve proteins differing in pI by less that 0.05 pH units. The technique is rapid and nondenaturating and can be run preparatively as well as analytically. lEF is well suited for use at any stage of a preparative scheme, and it is particularly effective in the early stages of purification. Electrofocusing differs from conventional electrophoresis by using pH gradient gels. Either the pH gradient within the gel preexists if ampholytes covalently bound to acrylamide are used for casting gels, or the pH gradient is formed during electrofocusing if mobile carrier ampholytes are used. This chapter describes isoelectric focusing using mobile ampholytes. During the experiment the carrier ampholytes arrange according to their isoelectric points, and a pH gradient develops.