Preparative-scale fractionation by isoelectric trapping under nondenaturing conditions: Separation of egg white protein isoforms on a modified Gradiflow unit

Abstract

pH-biased isoelectric trapping was used to separate proteins from egg white at the preparative level (80 mg), into discrete protein fractions based on isoelectric point. The problems of isoelectric precipitation that are common for the separation of complex protein mixtures under isoelectric conditions were mitigated by using single-component isoelectric buffers within the sample separation compartments. This combined with the mild process conditions of the Gradiflow unit that was modified for binary isoelectric trapping separations, ensured that biological activity was maintained. This was verified by measurement of the trypsin protease inhibitory activity of the extract and separated fractions. Furthermore, the high resolving power of this system under preparative conditions was demonstrated by separation of three protein isoforms using isoelectric membranes with differences of 0.025 pH units from each other.