The rate zonal separation of organelles from dilute suspensions: the problem of a large sample volume

Transport vesicles that deliver proteins to the cell surface can be isolated by incubating cells that have been permeabilized by mechanical or chemical techniques in a high-K medium containing an ATP regenerating system. The vesicles released from permeabilized cells are, however, obtained as a very dilute suspension in the incubation solution. This presents a problem for the preparative separation of specific populations of vesicles by velocity sedimentation, because the small sample volume capacity of traditional glycerol or sucrose velocity gradients requires that the vesicles first be concentrated by sedimentation or that very small amounts of vesicles be loaded onto a gradient. We have addressed the problem of the loss of zonal resolution produced by the loading of large sample volumes, and we propose that high-viscosity Ficoll gradients can be used effectively to restore the resolution of zones when substantially larger sample volumes of dilute suspensions must be loaded onto velocity gradients.