A method for rapid fractionation of particulate systems by gradient differential centrifugation.

IPIT initial experiments on the fractionation of mitochondria into subparticulate constituents, it became apparent that for isolation of the particles, a method more satisfactory than the customary methods of differential centrifugation was needed. The subject of this report is a simple and rapid method for obtaining relatively uncontaminated preparations of particulate cell components by gradient differential centrifugation, in quantities sufficient for further subfractionation. The method depends on the use of a centrifuge distribution head that allows rapid construction of liquid density gradients. Although we have been primarily concerned with the isolation of mitochondria free of nuclei and small particles, the method is applicable, with suitable modification, to a variety of particulate systems. The resolution of suspensions of particles, such as tissue breis, into fractions of relatively uniform size and density is greatly improved if the centrifugation is accomplished in continuous density gradients [l, 2, 4, lo]. This is because centrifugation anomalies that lead to mixing and cross contamination are largely eliminated [l, 21. The literature describing the experiences of several workers in adapting gradient differential centrifugation to various systems is reviewed by Anderson [2] and Svensson et al. [19]. Papers not mentioned by these authors are by Weber [22], who describes the separation of the particulate components of Xenopus eggs in density gradients, and by Thomson and Klipfel [20], who relate enzymatic activity to the size of particles isolated by gradient differential centrifugation from liver breis. Cardinal difficulties with previous methods of gradient differential centrifugation have been (a) the time required to produce the gradients and(b) the small volume of brei fractionated per tube. In the method previously described by Anderson [a], for example, 2-3 ml of material could be fractionated per tube, and the filling of each required 90 minutes.