Isolation of functionally distinct endosome subpopulations by free-flow electrophoresis

We have described a novel analytical procedure for the one-step purification of endosome subpopulations based on their differing electrophoretic mobilities when subjected to free flow electrophoresis. This procedure offers several advantages over existing schemes for endosome purification. Highly enriched endosomes are recovered in high yield and remain intact, facilitating biochemical and functional analysis of endosome subpopulations. It is rapid, requiring less than 2 hours to complete, and highly reproducible, thus facilitating detailed kinetic analysis of ligand transport through endosomal subpopulations. This technique can be easily scaled up to any degree enabling preparation of large quantities of early and late endosomes for further biochemical analysis and for antibody production. Our results have demonstrated the existence of at least two functionally and biochemically distinct, but kinetically related, subpopulations of endosomes.