Another
Density Gradient Fractionator Density gradient centrifugation is a frequently used procedure to separate subcellular components. In its simplest form the density gradient containing the sample is removed and collected drop by drop through a hypodermic needle piercing the bottom of the plastic centrifuge tube. Numerous methods have been proposed to improve the efficiency and ease of sample removal from density gradients but suffer from certain disadvantages. For example, time and expense may be annoyingly large as some procedures require such manual manipulations as drop-counting and destroy the centrifuge tube in the process. More important, in procedures which collect samples through a hole in the bottom of the tube, artifacts may occur when material which has pelleted to the bottom breaks free during fractionation of the gradient. Furthermore, because the centrifugal field is not strictly linear but fans out from a focus at the center of the rotor head, some material will be sedimented onto the walls of the tube during centrifugation.
As the meniscus of the gradient descends, it tends to wipe off some of this material and a false peak is observed.
This communication describes an inexpensive density gradient fractionator which does not necessitate the destruction of tubes, is easily coupled to automatic analyzing equipment and can accommodate any size of centrifuge tube used in a Beckman Instruments Company preparative ultracentrifuge.
An exploded view of the apparatus is shown in the drawing in Fig. 1. Solutions are introduced and withdrawn from the centrifuge tube via two thin-walled stainless-steel tubes of the type used for syringe needles. The two tubes, one inside the other, are silver soldered as a unit to a roundhead brass machine screw drilled through the center. A brass collar, threaded on both ends, holds the above-modified screw and a Beckman Instrument Company centrifuge tube cap. A larger collar fits over the inner brass collar and each collar is silver soldered to a blade of a hemostat such that when the hemostat is locked closed, the centrifuge tube cap tightly grips the tube containing the density gradient.
Because the tube is supported wholly by the centrifuge tube cap, the progress of elution of visible bands can be easily followed and, if necessary, the tube can be surrounded by a beaker containing ice in order to maintain the gradient at a low temperature during collection.