The ‘B’ form of the exo-β-N-acetyl-d-glucosaminidase from pig epididymis was purified by a new systematic strategy to yield a preparation with a specific activity at least equal to that obtainable by an existing empirically derived procedure. The new strategy is essentially a sequence of three carefully chosen steps consisting of an initial fractionation of the constituent proteins according to molecular size, followed by an ion-exchange step designed to select out proteins with closely similar electric-charge properties to those of the protein of interest, and a final high-resolution step dependent on the isoelectric points of the residual proteins. Gel isoelectric focusing itself, or as an element in the technique described by Leaback & Robinson (1974) for the separate display of the molecular size and electric-charge characteristics of proteins, played an important part in the choice of the experimental conditions used in the new strategy, and also in monitoring the progress of the purification.
High Resolution EB Lithography on Organic Resists: Molecular Size Effect.
