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tissue fractionation

# Differential Centrifugation

If you had sufficient time and a vibration-free environment, you could patiently wait and the force of gravity would bring most suspended particles to the bottom of a centrifuge tube. The smallest particles would probably stay in suspension due to brownian motion, and most macromolecules would be uniformly distributed because they would be in solution rather than suspension. I don't know about you, but I don't have the kind of patience needed in order to rely solely on gravity for separation of solid from liquid components. Besides, for practical purposes the pellet you obtained would be way too easily disrupted for effective separation of solid material from supernatant. Gravity would not be a terribly effective way of separating suspended materials based on size or other characteristics.

## Describing centrifugation conditions

When decribing a centrifugation run in materials and methods, it is seldom necessary to report more than the force, time, and temperature of centrifugation. The required speed (rpm) depends on the centrifuge and rotor used, which will vary from one lab to the next. Thus it is seldom relevant to report the make of centrifuge,type of rotor, or speed.

## The centrifugation process

Centrifugation produces a centripetal force that can be many hundreds or thousands of times the force of gravity, thus speeding up the process considerably. The greater the number of revolutions per minute (RPM), the greater the force of gravity. The usefulness of centrifugation in cell fractionation would be limited if all we could do is drive suspended particles to the bottom of a tube. However, investigators are able to control the size of particles that are brought down, thanks to the physics of particles in suspension.

In a suspension of round particles of equal density but different diameters, the force that drives a given particle to the bottom is equal to its mass times the applied acceleration. The volume of the particle is a function of its radius, and its mass is equal to its volume times its density coefficient, which is a constant. The volume of a sphere is equal to 4/3 times pi (a constant) times the cube of the radius. For a suspension of spherical particles of equal densities under a specific set of conditions, the only variable that determines the force on a given particle is its radius.

The resistance to movement through a solution is proportional to that part of the surface area that pushes through the medium. For particles of similar shape, smaller particles encounter less resistance than larger ones. Since the surface area of a sphere is 4 times pi times the square of the radius, and 4 times pi is a constant, then for spherical particles of equal composition, the only variable that determines resistance under a given set of conditions is the radius of the particle.

Driving force increases proportionally to the cube of the radius. Resistance to movement increases proportionally to the square of the radius. It isn't difficult to see that as the radius of a particle increases, its tendency to approach the bottom increases as well. Add a significant amount of 'drag,' and the gravity experiment that has been attributed to Galileo doesn't work so well, after all. Since large particles sediment more rapidly than small particles, an investigator can separate large from small organelles, cells, etc. simply by controlling the time and rpm of a centrifuge run.

## Fractionation by differential centrifugation

For a typical cell homogenate, a 10 min. spin at low speed (400-500 x g) yields a pellet consisting of unbroken tissue, whole cells, cell nuclei, and large debris. The low speed pellet is traditionally called the nuclear pellet. A 10 min. spin at a moderately fast speed, yielding forces of 10,000 to 20,000 x g brings down mitochondria along with lysosomes and peroxisomes. Therefore the second pellet in the traditional cell fractionation scheme is called the mitochondrial pellet.

Further cell fractionation by differential centrifugation requires the use of an ultracentrifuge. Such an instrument is designed to spin rotors at high angular velocities, to generate very high g forces. The air must be pumped out of the chamber in order to avoid heat buildup due to air friction. In fact, many rotors that are designed for an ultracentrifuge are not even built aerodynamically, since they are spun in a vacuum. A one hour high speed ultracentrifuge run that generates a force on the order of 80,000 x g yields a microsomal pellet. Microsomes include fragments of membrane, including cell membrane and endoplasmic reticulum. Membrane fragments form vesicles when disrupted in an aqueous medium, so examination would reveal numerous membrane vesicles of various sizes. The vesicles themselves can be separated on the basis of density, due to varying protein content. But that's a subject for another document.

Spin for several hours at 150,000 x g or so, and you can bring down ribosomes and even the largest of macromolecules. The supernatant that remains consists of soluble components of the cytoplasm, including salts, small macromolecules and precursor molecules, and dissolved gases.