Human lysosomes can be purified from diploid skin fibroblasts by free-flow electrophoresis

Abstract

Human diploid fibroblasts underwent lysis when resuspended in isotonic sucrose. After preparation of a granular fraction by differential centrifugation, lysosomes were almost completely purified by free-flow electrophoresis. Electron micrographs of the final fraction of fibroblast lysosomes (about 25-fold purified) showed mostly secondary lysosomes having an appearance identical to those in intact cells. There was no indication that the purification steps selectd for any lysosomal subpopulation. The lysomal nature of the isolated organelles was confirmed by cytochemical staining for acid beta-glycerophosphatase. As judged by a 92% structure-linked latency of beta-N-acetylglucosaminidase in the final fraction, the structural integrity of the isolated lysosomes was unaffected by the purification procedure.