Separation of endosomes by aqueous two-phase partition and free-flow electrophoresis

We have developed two endosome models to evaluate the separation of endosome populations by aqueous two-phase partition. In the first model, bovine kidney endosomes were used. In the second model. HeLa endosomes were identified in homogenates by means of a latent drug-(capsaicin-)inhibited NADH oxidase (NOX). Endosomes were first isolated by aqueous two-phase partition. To separate early and late endosomes, the endosomes were incubated with ATP to acidify the endosome interiors by activating a proton-translocating ATPase. Thus far, we have been able to resolve the early and late endosomes from any source only by preparative free-flow electrophoresis and not by phase-partition. Previous studies have shown that gravitational forces may be important for separation of endosomes by phase partition. Low-speed centrifugation (< or =12.5 g) during phase resolution altered the activity of the latent NADH oxidase used as a marker for HeLa cell endosomes.