Free-flow electrophoresis: preparative applications to cell-free analyses of exocytotic membrane traffic.
Preparative free-flow electrophoresis provides a useful adjunct to conventional centrifugation methods with important applications to isolation of exocytotic and endocytotic compartments. With rat liver, Golgi apparatus subfractionation into cis, trans and median compartments is achieved. Simultaneous isolation of plasma membrane-, endoplasmic reticulum-, Golgi apparatus, mitochondria-, nuclear envelope- and possibly lysosome-enriched fractions is obtained with homogenates from cultured cells. Other applications include subfractionation of plasma membrane vesicles of differing charge (including separation of inside out and right side out vesicles), purification of endoplasmic reticulum, isolation of tonoplast (vacuole membranes) from plants and separation of Golgi apparatus membranes and dictyosome-like structures of spermatocytes. Of particular importance to cell-free systems for study of membrane trafficking are recent successes with preparative free-flow electrophoresis to obtain preparations of transition vesicles (those with nap-like but not clathrin-derived coats, coming from endoplasmic reticulum and thought to be responsible for delivery of materials to cis Golgi apparatus elements).
Morré
D J
DJ
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, IN 47907.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D002478:Cells, Cultured; D004586:Electrophoresis / Q000379:methods*; D005089:Exocytosis; D006056:Golgi Apparatus / Q000032:analysis / Q000648:ultrastructure; D008099:Liver / Q000032:analysis / Q000648:ultrastructure; D008854:Microscopy, Electron; D051381:Rats; D013347:Subcellular Fractions