THE RATE ZONAL SEPARATION OF ORGANELLES FROM DILUTE SUSPENSIONS - THEPROBLEM OF A LARGE-SAMPLE VOLUME

Transport vesicles that deliver proteins to the cell surface can be is olated by incubating cells that have been permeabilized by mechanical or chemical techniques in a high-a medium containing an ATP regenerati ng system, The vesicles released from permeabilized cells are, however , obtained as a very dilute suspension in the incubation solution, Thi s presents a problem for the preparative separation of specific popula tions 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 t hat very small amounts of vesicles be loaded onto a gradient, We have addressed the problem of the loss of zonal resolution produced by tile loading of large sample volumes, and we propose that-high-viscosity F icoll gradients can be used effectively to restore the resolution of z ones when substantially larger sample volumes of dilute suspensions mu st be loaded onto velocity gradients. (C) 1998 Academic Press.