Current biochemical characterization for cholesterol- and glycolipid-rich membrane microdomains largely depends on analysis of detergent-resistant membranes (DRMs). In the present study, we succeeded in separation of DRMs of similar density-based on their electrical charge using free-flow electrophoresis (FFE). After crosslinking of B cell receptor (BCR), mouse splenic B cells were lysed with 1% Brij-58 and the resulting lysate was subjected to sucrose density gradient ultracentrifugation. The low-density fraction that recovered a part of DRMs containing IgM together with those enriched in GM1a, the Src family protein tyrosine kinase Lyn, and the alpha subunit of inhibitory heterotrimeric GTP-binding protein was further resolved by FFE. FFE separated the former into more cathodally deflected fractions than the latter. In addition, FFE revealed an anodal shift of DRMs containing a transmembrane protein CD38 upon BCR-crosslinking. The results demonstrate the effectiveness of FFE for the charge-based separation of DRMs. (C) 2004 Elsevier Inc. All rights reserved.