We describe a new method for large-scale separation of membrane-associated biomolecules using a packed bed of bilayer-coated particles as an electrophoretic medium. The basic strategy is as follows. First, solid particles coated with fluid lipid bilayers and those coated with fluid lipid bilayers containing the sample to be separated (lipids or membrane proteins) are packed in a column so as to form a "sample plug." Next, electrophoretic separation of the sample components is carried out by applying a dc electric field along the column. Finally, the separated fractions are collected by taking the particles out from the column. The above strategy is based on the continuity of the bilayer throughout the column-the bilayers on adjacent particles are fused together under a densely packed condition. We demonstrate proof-of-concept experiments using fluorescently labeled lipids as the sample. This method will provide an easy way for production of functional particles coated with membrane-associated biomolecules.