Rate-zonal centrifugation

Fig. 11. (a) Differential centrifugation (pelleting), where time 1 < time 2 < time 3 < time, (b) Rate zonal separation in a swinging-bucket rotor, where tube 1 represents the density gradient solution, tube 2 the sample plus the gradient, and tube 3 the separation of sample particles under a centrifugal force, where... 

In another approach subcellular fractionation of human PMNs, stimulated in vitro with phorbol myristate acetate or opsonized zymsan, was performed by rate-zonal centrifugation in sucrose gradients By varying the centrifugal force the NADPH oxidase could be separated from the activities of enzymes in the lysosomal granules but was similar to the distribution of markers for the plasma membrane, aryl phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. 

Rate-zonal In rate-zonal centrifugation the sample is layered at the top of a centrifuge tube... 

Proteins with large differences in molecular mass can be separated by rate-zonal centrifugation using a gradient of a dense material such as sucrose. 

The zonal rotors will not be described in detail here, since they are habitually chosen for large volume preparative protocols (typical capacities are 300-1700 mL), mainly using rate-zonal centrifugation. Basically, a zonal rotor consists of two half-cylinder sections, which screw onto one another. The sample is poured inside the rotor without the use of tubes. 

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). 

Figure 13.7. Comparison between (a) rate-zonal and (b) isopycnic centrifugation. Note that the gradient used in rate-zonal techniques is shallow, and if the centrifugation continues, the particles pellet. The diffusion process causes band broadening in rate-zonal centrifugation, but this is not evident when the particles are focused using isopycnic centrifugation.

In rate-zonal centrifugation the conditions for sensitive biological particles are gentle (low osmotic pressure, g forces, and viscosity) and typically the separation is faster than isopycnic separations. Applications include the separation of proteins and nucleoproteins, like ribosomes. 

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 ... 

Figure 18.4 Rate zonal centrifugation in a preformed liquid density gradient.

Figure 3 Rate-zonal separations in a swinging-bucket rotor. (A) Centrifuge tube fiiied with density gradient soiution, (B) sample applied to top of gradient, and (C) under centrifugal force, particles move at different rates depending upon their mass. Courtesy of Beckman Coulter, Inc.)...

Rate-zonal centrifugation may be used for preparation of larger amounts of DNA nanostructures. 

Figure 10.10 Rate-zonal centrifugation. (A) Sample layered over a continuous density gradient. (B) Position of zones due to different size of particles at the termination of centrifugation. Note that separation is due to differences in size and shape.

Table 10 1 Significant differences between rate-zonal and Isopycnic centrifugation...

A gradient matericd should meet several requirements. There is no ideal all-purpose gradient materia). Sucrose is used routinely for rate-zonal centrifugation, while cesium chloride may be the usual choice for isopycnic centrifugation. Apart from allowing the desired type of separation, the gradient material should have the following properties ... 

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