Recent studies have demonstrated that membrane ultrafiltration can be used to separate proteins with very similar size, but these investigations have been performed with model binary mixtures. This study examined the use of a two-stage tangential flow filtration system for the purification of both alpha-LA and beta-LG from whey protein isolate. Separation was achieved using 100 and 30 kDa membranes in series, with the buffer concentration and filtration velocity for each stage adjusted to give optimal separation. Two purification strategies were examined: one using the 100 kDa membrane followed by the 30 kDa and one with the reverse order. In both cases, the alpha-LA purification was greater than 10-fold at 90% yield. The recovery of beta-LG was more challenging since it was obtained in the permeate from one stage and the retentate from the other. The results provide important insights into the development of staged membrane systems for the purification of complex protein mixtures. (C) 2003 Elsevier B.V. All rights reserved.