Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.