Sedimentation rate-zonal

Sedimentation Rate-zonal sedimentation, Equilibrium sedimentation,... 

Method Density gradient. Rate-zonal. The rate-zonal method is one of six addressed by SpinPro. The other methods are differential, differential-flotation, discontinuous, isopycnic, and 2-step isopycnic. These methods differ dramatically in their set up, principles of operation, and expected results. The rate-zonal method is described here briefly so that the recommendations to follow can be appreciated. Prior to the run in a rate-zonal method, a gradient material is introduced to the rotor tubes in steps of increasing density from the top to the bottom of the tube. The sample to be separated is layered, as a thin band, on the top of the gradient. As the run begins, each component in the sample moves toward the bottom of the tube. Some components sediment faster than others. This fact is the basis for the separation. If the run parameters are appropriate, the components will form separate bands within the gradient. At the conclusion of the run, the band representing the component of interest can be removed from the tube. 

Examination of the groupings of Table 7.2 verifies most of the more or less traditional associations between separation methods chromatography with countercurrent distribution and distillation, electrophoresis with rate-zonal sedimentation, isoelectric focusing with isopycnic sedimentation, re-... 

Peak capacity nc, like resolution, can be calculated in a straightforward manner for both isoelectric focusing and isopycnic sedimentation. However, it is most interesting to cast ne in a form such that these steady-state methods can be related to the analogous transient methods, namely, zone electrophoresis and rate-zonal sedimentation. To do this we substitute a from Eq. 8.42 into the basic peak capacity expression nc = L/4o-, Eq. 5.59. Then using D = 9177/, we get... 

In rate-zonal centrifugation (or velocity sedimentation) the sample is loaded as a thin layer on the top of a density gradient medium. During centrifugation, the sample separates into bands, and the particles are separated on the basis of their different sedimentation coefficients (s). For biological particles, the coefficient s is mainly related to the size of the particles. This process is illustrated in Figure 13.7(a). 

A number of techniques can separate proteins on the basis of their differences in mass. Describe the use of two of these techniques, centrifugation and gel electrophoresis. The blood proteins transferrin (MW 76 kDa) and lysozyme (MW 15 kDa) can be separated by rate zonal centrifugation or SDS polyacrylamide gel electrophoresis. Which of the two proteins will sediment faster during centrifugation Which will migrate faster during electrophoresis ... 

Rate-zonal separations It can be used to separate particles of similar density according to size or to separate particles of different density and size as a function of their sedimentation coefficient, s. In its simplest form, the sample mixture is layered in a narrow band on top of a preloaded, homogeneous medium as shown in Figure 3. 

After conducting rate zonal sedimentation of RNA in a CsCl gradient, the worker finds out that none of the regions in the tube absorbs at 260 nm. Why should that be so ... 

Density-gradient centrifugation, although not readily scalable, is a popular technique for final purification of viruses [Poison, 1993], Either rate-zonal centrifugation, where the separation is based on differences in sedimentation rate, or isopycnic equihbrium centrifugation, where the separation is based solely on density differences, is used. Further details on these techniques can be found in Dobrota and... 

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