Buoyant density centrifugation,

Figure 5.5 Some methods for the separation and isolation of homogeneous cell populations from tissue fragments. The tissue, composed of three cell types (O, , A), is disrupted and a solution of single cells obtained. The cell types are shown being separated by (/l) buoyant density centrifugation, (0) selective cellular lysis, (C) affinity chromatography, and (D) magnetoseparation.

The small particles of plasma lipoprotein, which carry triacylglycerols, can be separated according to their buoyant densities by centrifugation. They have been classified into five groups of increasing density but smaller size as chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density (LDL), and high density lipoproteins (HDL) (Table 21-1 and Fig. 21-2). Each lipoprotein particle contains one or more apolipoproteins (Table 21-2), whose sizes vary from the enormous 4536-residue apoB-100 to apoC-II and apoC-III, each of which contains just 79 residues73 and the 57-residue apoC-I.7b... 

Combine the required amount of CsCl stock solution, 1 pg of undetermined DNA, 1 fig of Micrococcus luteus reference DNA (buoyant density 1.7311 g/ml),6 and bring to a final volume of 500 fi with distilled water. Load centrifuge cells. Centrifugation is generally for 20 hr at 44,000 rpm.17... 

The lipoprotein density that should be employed in Eq. (2) is the buoyant density, which is the density at which the sedimentation coefficient is equal to zero. The buoyant density was obtained experimentally by measuring the sedimentation coefficient at several solvent densities and extrapolating to zero. Kahlon et al. (1982) have shown that the buoyant densities, which they call the a densities, vary with the rotor speed of the centrifuge, reflecting the different compressibilities of water and lipid. In order to convert the lipoprotein density, as determined from its composition, to the buoyant density at a rotor speed of 52,640, the data of Kahlon et al. (1982) were used calculate a correction factor of 0.0016 g/ml, which was added to the compositional densities. The values of buoyant densities listed in Table 11 have been calculated by adding 0.0016 g/ml to the density values determined from their compositions. 

In some cases it is possible to purify conjugates by density gradient centrifugation, provided that the buoyant densities or sedimentation coefficients are sufficiently different (Mannik and Downey, 1973 Ford et al., 1978). 

Native apoferritin can be prepared by a number of centrifugal methods. Density gradient centrifugation (62) in isopycnic gradients of CsCl or in sucrose have been used to fractionate native ferritin according to its iron content. The native apoferritin sediments with a buoyant density of 1.23, whilst full ferritin has a density in excess of 1.8. Typically using an SW 27 rotor 100 hr are required at 25.000 rpm with a CsCl... 

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