Zonal rotors

Zonal techniques may be used for the separation of a wide range of particles and macromolecules, e.g. mitochondria, nuclei, ribosomes and proteins. The technique may be used for bulk preparative work using a zonal rotor which is filled with a solvent gradient while running at a slow speed. The sample is similarly introduced and the rotor speed is then increased to the desired value. After centrifugation is complete, the contents are drawn off while the rotor is running slowly by displacing them with a more dense solution. 

Specialty rotors permit ordinary botde centrifuges to achieve some of the results previously considered possible only in ultracentrifuges. A modified zonal rotor, shown in Figure 9, permits collection of sediment using continuous addition of feed and discharge of centrate. 

Fig. 12. Dynamic loading and unloading of a zonal rotor, (a) Gradient is loaded while rotor is spinning at 2000 rpm (b) a sample is injected at 2000 rpm, followed by injection of overlay (c) particles separated when the rotor is running at speed and (d) contents are unloaded by introducing a dense solution at...

This involves the use of a special rotor and the adaptation of the centrifuge for constant flow as for the zonal rotor. This is expensive, but once installed, reproducible separations of 108-109 cells can be achieved in under an hour and cells of all sizes (rather than just the smallest) are obtained. It is particularly suited to the isolation of Gl-phase cells from suspension cultures for which mitotic detachment procedures are not applicable. The separation chamber is kite-shaped with the buffer solution entering at the acute point on the rim of the centrifuge and leaving at the obtuse point towards the centre of rotation (Fig. 11.2). Thus the centrifugal forces on particles within the chamber are countered by the centripetal flow of buffer and particles come to equilibrium within the chamber. When equilibrium has been reached, the samples are pumped out, by increasing the rate of buffer flow, and collected. Meistrich et al. (1977) obtained 3 fractions of L-P59 mouse fibroblasts which were over 90% Gl-phase, 70% S-phase cells and 60% G2 + M-phase cells, respectively. 

While there are many practical problems in realizing the theoretical power of sedimentation, major advances have accompanied the development of the disc centrifuge and of the zonal rotor [43-45]. 

We found earlier that transient (nonsteady-state) sedimentation required density gradients to stabilize against convection. Isopycnic sedimentation relies on density gradients not only for anticonvective purposes but also as the secondary gradient needed to establish steady-state conditions. The difference in the two cases is found in the magnitude of the density gradient and in the degree to which components are allowed to approach their steady-state condition. The equipment is similar the zonal rotor developed by Anderson is used for isopycnic as well as transient zonal separations [45]. 

Although the use of zonal rotors is somewhat more involved than conventional swinging bucket rotors it is important to emphasize that the end result is the same. An example of this is shown in Figure 9-32, which depicts the elution profile of a stepped sucrose gradient that was used to separate mitochondria, proplastids, and glyoxysomes. This profile is very similar to that shown in Figure 9-16A, but in that experiment the gradient volume was 54 ml. Here it is 1760 ml and accommodates the crude particulate fraction yielded from 1 lb of fresh tissue. 

Figure 9-32. Separation of crude particulate fraction from castor bean endosperm using a B-XV zonal rotor.

The details of these methods are not covered in this manual but a further introduction can be found in, for example, An Introduction to Ultracentrifugation by Bowen (1970) and a great deal of detailed theory in Schachman (1959). Experimental techniques using zonal rotors are reviewed in a monograph edited by Anderson (1966) and some more recent developments are in Methodological Developments in Biochemical Separations and Methodological Developments with Zonal Rotors both edited by Reid (1972). 

Conn, H. J. (1946) Biological Stains, 5th Ed. Biotech. Publications, Geneva, N.Y. Cope, J. R. and H. R. Matthews (1972) In E. Reid, ed., Methodological Developments with Zonal Rotors (Longmans). 

Reid, E. (1972) Ed. Methodological Developments in Biochemical Separations and Methodological Developments with Zonal Rotors (Longmans, London) (in press). Reunders, L., P. Sloop, J. Sival and P. Borst (1973) Biochim. Biophys. Acta 324,320. Reynolds, J. A. and C. Tanford (1970a) Proc. Nat. Acad. Sci. 66, 1002. 

There are four basic types of rotors the swinging-bucket, fixed-angle, vertical, and zonal rotors. The vertical rotor could be classified as a fixed-angle rotor, in which the angle is extreme, but most authors consider that they are different enough to consider separately. A detailed description of these four types of rotors and their specifications can be found in Rickwood.5... 

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. 

Fig. 2. The subfractionation of the P2 fraction from rabbit cortex on a discontinuous sucrose gradient in a zonal rotor after the initial removal of myelin. The BXIV rotor, capacity 650 ml, was used and...

We are grateful to the Multiple Sclerosis Society of Great Britain for funds with which the zonal rotor and the labelled choline were purchased. We would also like to thank Mr. J. Candy for examining some fractions by fluorescence microscopy and Professor P. B. Bradley for his interest in the work. 

Spanner, S. and Ansell, G. B. (1971) Preparation of subcellular fractions from brain tissue. In Separations with Zonal Rotors, E. Reid (Ed.), Wolfson Bioanalytical Centre of the University of Surrey, Guildford, pp. V-3.1-3.7. 

Zonal rotors are bowls or cylindrical cavities equipped with a central core and attached vanes... 

Figure 7 Static loading and unloading of a zonal rotor with a reorienting gradient core. (A) Gradient loaded, light end first, with rotor at rest (B) sample solution layered on top of gradient (C) Rotor accelerated, layers reoriented under centrigufal foce (D) layers vertical, particles separated with rotor at speed (E) rotor decelerated, Layers reoriented (F) static unloading, contents displaced with air pressure, heavy end first. (Courtesy of Beckman Coulter, Inc.)...

Continuous zonal rotors These rotors are similar to those designed for batch separation but with a larger diameter core providing a different flow pattern as illustrated in Figure 12. These rotors are best suited for low-concentration, high-volume samples. Applications include purification of viruses from tissue-culture media, harvesting bacteria, or separating fine-clay particles from water. 

A variant of this procedure is the use of a zonal rotor which, due to its much larger area of the sample cavity, allows the separation of relatively large amounts of lipoproteins. Patsch et al. described three subfractions in HDL2 and five subfractions in HDL3 [4]. But, as in Anderson s study [2], there was considerable overlapping of fractions. 

Basically, rotors come in four varieties fixed-angle rotors, vertical tube rotors, swinging-bucket rotors, and zonal rotors. The first three rotors are discussed below. Zonal rotors will be discussed In a later section. 

Attempts have been made to minimize wall effects by using a sector shaped cell in the swinging-bucket rotor. However, the best way to minimize the wall effects is to use a zonal rotor. The discussion of zonal rotor is deferred to a later section as an understanding of density gradient centrifugation is essential to understand its working. 

Figure 10.14 (A) Sector shaped cell of a zonal rotor. Wall effects do not pose a problem. (B) Design of a zonal rotor (B-XV). Note the four veined core. Arrows indicate ports at the base ends of the veins.

Figure 10.14B shows the design of a zonal rotor. As evident from the figure, the zonal rotor is more or less a flattened sphere which has its Interior subdivided into four equal quadrants... 

Figure 10,15 Cross section of one quadrant of a zonal rotor showing (Q the special holes drilled into the septa for gradient loading, and (W sample port G-Channels through which gradient is loaded S-f Sample port...

Again unlike conventional methods (in which the most dense solution is poured first) the gradient in a zonal rotor is established by pumping the material of the lowest density first. In the... 

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