Centrifugation 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. 

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]. 

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. 

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. 

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... 

Boone et al. (1968) centrifuge cells through a discontinuous 10-20% Ficoll gradient made up in Eagle s minimum essential medium modified for suspension (i.e. lacking calcium and bicarbonate and containing 10 times the normal phosphate concentration). They use an A-1X zonal centrifuge rotor and spin for 1 h at 1000 r.p.m. at 20°C, and obtained clear separation of different cell types (HeLa and rabbit thymocytes). 

Figure 4.15. Zonal Centrifugation. The steps are as follows (A) form a density gradient, (B) layer the sample on top of the gradient, (C) place the tube in a swinging-bucket rotor and centrifuge it, and (D) collect the samples. [After D. Freifelder, Physical Biochemistry, 2d ed. (W. H. Freeman and Company, 1982), p. 397 ]...

The separation of the components of the myelin-free crude mitochondrial fraction of whole brain tissue in centrifuge tubes is compared with a separation by zonal centrifugation. On a shallow, step-wise gradient of 0.8-1.7 M sucrose in a BXIV rotor of 650 ml capacity, it was possible to obtain lysosomal, mitochondrial, synaptosomal and plasma membrane fractions after spinning for 2 h at 67,000 x g. These fractions were characterised by enzyme markers and other means. At least two synaptosomal populations could be clearly separated, one of which could actively take up a-methylnoradrenaline. Some preliminary studies on the uptake of [ A/e-14C]-choline into sub-cellular components after intracerebral injection are also described. 

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.)...

Barradas RG, Donaldson GJ, Shoesmith DW (1973) Double layer studies of aqueous sodium citrate solution at the mercury electrode. J Electroanal Chem 41 243-258 McDonald DM, Hsu HW (1972) Transport phenomena in zonal centrifuge rotors. VI. Concentration-dependent diffusivities of potassium citrate and potassium tartrate in aqueous solutions. Separ Sci 7 491-503... 

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