Cell fractionation procedures involving differential sedimentation followed by resuspension of pellets and isopycnic centrifugation are very difficult to apply to the small amounts of material available from tissue culture cells. We have explored the possibility of successive differential and isopycnic sedimentation in a zonal rotor using a short viscosity barrier for the differential sedimentation. The marker enzymes used were cytochrome oxidase, acid phosphatase, catalase, and 5'-nucleotidase. The results of these procedures are compared to the results of one-step isopycnic separations in gradients of sucrose and Stractan. The Stractan gradient was much more effective than the sucrose gradient in separating the marker enzymes from the proteins of a postnuclear supernatant, but neither type of gradient could significantly purify the marker enzymes one from another. A two-step procedure using a viscosity barrier was effective in separating particles carrying catalase from the other marker enzymes assayed and from most of the protein. A three-step procedure resulted in similar purification of mitochondria. Modification of barrier composition and centrifugation times would probably result in further improvement of separations according to individual requirements for yield, purification, and freedom from specific contamination by other subcellular particles.
Subcellular fractionation by centrifugation has proven a powerful tool in many areas of cell biology. Rat liver is the tissue that was first fractionated (1) and remains today the tissue that is most frequently fractionated. However, the technique has been applied to a wide variety of other cells and tissues, generally with methods that were pioneered in fiver fractionation. Usually the subcellular particles are separated into size groups by differential centrifugation in preparative angle-head rotors, and then these fractions from differential centrifugation are further subfractionated by isopycnic centrifugation in density gradients of various compositions. These isopycnic centrifugations can be performed either in swinging-bucket rotors or zonal rotors. The convenience of operation of zonal rotors has made possible fractionation techniques in which particles separated in one gradient may then be further frac-