AbstractWe have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high‐resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis1993,14, 1011–1018). Isoforms of bovine β‐lactoglobulin, human apo‐transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and