Trennung biologischer Partikel durch Zentrifugation im Dichtegradienten

Abstract

Density gradient centrifugation is a very effective procedure for both analytical and preparative separation of particles and macromolecules.
The principle is quite simple. The mixture of particles is suspended in a medium of light specific weight and layered on top of a preformed density gradient. Since the components form discrete zones, when centrifuged, the procedure is also called zonal centrifugation. Separation is primarily obtained according to the sedimentation rate (rate zonal separation), which depends predominantly on the particle size.
If a density gradient is used, which covers the complete density range of all fractions, particles reach level of equal density (buoyant density) after a sufficient period of time (isopycnic zonal separation).
In ultracentrifuges at sufficiently high speeds the density gradient does not need to be preformed, but is formed by the centrifugal field itself.
Unlike differential centrifugation, zonal methods allow the quantitative recovery of the separated constituents. Zonal separations in centrifuge tubes with swinging-bucket and fixed-angle rotors and in sector cells have been used for the determination of physical characteristics, such as molecular weight, sedimentation coefficients, density and heterogeneity. Because of the low capacity, however, such experiments have more analytical than preparative significance. For preparative work zonal rotors, consisting of a bowl-shaped vessel, are applied. With these rotors, which combine a large capacity with the freedom from mechanical disturbances and wall effects, a great number of biological particles and macromolecules have been isolated, such as nuclei, mitochondria, polysomes, ribosomes, viruses and macroglobulines. Zonal rotors for continuous flow operations allow the collection and purification of particles from very large quantities of medium.