Zonal Centrifugation: Dynamically Unloaded, Zonal Rotors and Reograd Zonal Rotors

Density gradient centrifugation using tubes is the most widely employed technique for separating cells and cell organelles and for isolating cellular macro­molecules.

However, although it is one of the cell biolo­gist’s most valuable tools, it is not without disadvan­tages, as the amount of material that can be fractionated in a single tube is so small.

When large quantities of sample must be fractionated (to isolate sparse organelles such as lysosomes or peroxisomes), a very large number of tubes and gradients is needed. Much larger quantities of sample may be fractionated using zonal rotors.

A zonal rotor consists of a large cylindrical chamber subdivided into a number of sector-shaped compart­ments by vertical septa (or vanes) that radiate from the axial core to the rotor wall.

The entire chamber is used during centrifugation and is loaded with a single density gradient, each sector-shaped compartment serving as a large centrifuge tube. The large chamber capacity of these rotors (typically 1 and 2 liters) elimi­nates the need for multiple runs and multiple density gradients.

Two basic forms of the zonal rotor are used for tissue fractionation:

(1) Dynamically unloaded (rotating-seal) rotors and

(2) Reorienting gradient (“reograd”) rotors.

The similarity in appearance of these rotors (Fig. 12-12) is misleading, as the basic principles of operation are substantially different.

Dynamically Unloaded Zonal Rotors:

Operation of a dynamically unloaded zonal rotor is de­picted schematically in Figure 12-13. Two fluid lines connect the center and edge of the rotor chamber with a rotating-seal assembly that permits loading the den­sity gradient (and sample) into the rotor while the ro­tor is spinning. The center fluid lines open into the ro­tor chamber through the core section, and the edge lines pass radially through each of the septa and open at the rotor wall.

The zonal rotor is filled with density gradient while rotating at low speed. The light end of the gradient is loaded first through the edge lines and is followed by the denser solution (Fig. 12-13a). The dense end of the gradient gradually displaces the lighter fluid toward the core of the rotor. Addition of a dense “cushion” forces some of the light end of the gradient out of the rotor (Fig. 12-13b).

The sample to be frac­tionated is introduced through the center lines, thereby displacing some of the cushion out of the edge lines (Fig. 12-13c). Additional light fluid (called over­lay) is then pumped into the center line to push the sample clear of the core region (Fig. 12-13d).

Now the upper (stationary) portion of the seal assembly is re­moved and the rotor is accelerated to a higher speed for separation of the particles in the sample (Fig. 12- 13e). The separated particles form a series of concen­tric cylindrical zones in the rotor bowl. Following par­ticle separation, the rotor is decelerated to a lower speed, the static seal is reinserted, and the entire gra­dient is displaced through the center lines by pumping dense fluid through the edge line (Fig. 12-13f). The eluting gradient may be monitored and collected in tubes for subsequent analysis.

Reograd Zonal Rotors:

The operation of reograd zonal rotors is depicted In Figure 12-14. Unlike dynamically unloaded zonal ro­tors, the gradient and sample can be loaded into the reograd rotor either at rest through the septa lines (which communicate with the bowl floor) or while spinning using the core lines. Unloading is always car­ried out with the rotor at rest.

If the rotor is loaded at rest, then the gradient is reoriented from the vertical to the radial position during acceleration (Figs. 12-14a and 12-14b). During centrifugation, different parti­cles in the sample sediment to form a family of con­centric cylindrical zones in the radial gradient (Fig. 12-14fd), but during rotor deceleration, they are reori­ented to form horizontal layers (Figs. 12-14e, and 12- 14f). The density gradient and entrained particles are then withdrawn from the stationary rotor through the septa lines and collected as fractions for further anal­ysis.