Chapter 17 Endosome and Lysosome Purification by Free-Flow Electrophoresis

Abstract

Free-flow electrophoresis (FFE) has, for some time, been used in the separation of specific cell types, as well as proteins and larger multimeric complexes. In recent years, however, the technique has been applied to the fractionation of subcellular membrane-bound organelles. In this chapter the procedures that have been adapted or developed to isolate endosomes and lysosomes are described. In combination with Percoll density gradient centrifugation, FFE has been used to prepare endosomes, endosome subpopulations, and lysosomes from tissues and cultured cells. It is unlikely that FFE will become a one-step technique for the purification of subcellular organelles, but in combination with other fractionation procedures, it has a very powerful addition to the approaches that can be applied to cell fractionation. Significantly, FFE is likely to put less stress on organelles during fractionation and increase the yield of intact organelles, because the forces imposed on the organelles during brief transit through the electric field are likely to be considerably less than those imposed by centrifugation. The full potential of the technique remains, however, to be realized. © 1989 Academic Press Inc.