Figure 3.5 Svedberg Values in Percoll and Sucrose
Table 3.3 Physical characteristics of gradient media
Centrifuge the four tubes for the equivalent clearance of Beckman 30.2 rotor at 35,000 xg for 15 minutes at 20° C.
Pertoft and coworkers developed a synthetic, colloidal solution of polyvinylpyrrolidone coated silica, specifically designed for sedimentation centrifugation. This material is marketed under the trade name of Percoll. Table 3.3 give the characteristics of this medium, compared to several other density media, namely sucrose, metrizamide and Ficoll.
Of particular interest is the fact that during centrifugation in a fixed angle rotor, Percoll will spontaneously form linear gradients, the shape of which is dependent upon rotor speed and time of centrifugation. Thus, it becomes a simple matter to establish a linear density gradient.
Figure 3.5 demonstrates a comparison of Percoll fractionation of cellular components to sucrose fractionation. This figure also presents, a standard way to comare components, by defining the relationship of density to the sedimentation coefficient for a cell or organelle. Note, that lymphocytes, granulocytes and erythrocytes have very similar sedimentation coefficients, but can be separated on the basis of density. Organelle separations are much easier to accomplish on Percoll density gradients than on sucrose gradients.
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