High resolution density gradient electrophoresis of cellular organelles.

Abstract

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 x 2.2 cm) containing a 0-5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O-ring. The top circular platinum anode allows for upward electrophoresis (80-100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle-enclosed enzymes.