The objective of this study is to investigate a combination of ion exchange membranes and free-flow isoelectric focusing (FFIEF) technology for high-selectivity and high-flux protein separation, in which ion exchange membranes are used as the separation media. An FFIEF device has been designed and extensive experiments have been conducted to prove its effectiveness in enhancing the protein separation performance. Three types of membranes were employed in this work and they were commercial microfiltration (MF) ion exchange membranes, commercial neutral UF cellulose membranes, and home-made ultrafiltration sulfonated polysulfone (UF SPSf) ion exchange membranes. The protein separation results show that the home-made UF SPSf membranes have the superior selectivity and flux to other membranes. This is due to the fact that a stable pH gradient across the membranes as well as the interaction between the protein molecules and membrane surface plays an important role in the high-performance protein separation. By applying a semi-batch separation process and optimizing various experimental conditions, a high-purity (> 90%) and concentrated target protein is obtained at the permeation side of the home-made UF SPSf membranes with a high flux. (C) 2008 Elsevier Ltd. All rights reserved.