Tuesday, April 28, 2009

What is Density Gradient Centrifugation

Density gradient centrifugation are of two types:

1) Rate zonal centrifugation

2) Isopycnic centrifugation

Rate zonal centrifugation:
In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s). The run must be terminated before any of the separated particles reach the bottom of the tube. S is the sedimentation coefficient and is usually expressed in Svedbergs (S) units.

Isopycnic centrifugation:
In the isopycnic technique, the density gradient column encompasses the whole range of densities of the sample particles. The sample is uniformly mixed with the gradient material. Each of the particles will sediment only to the position in the centrifuge tube at which the gradient density is equal to its own density, and there it will remain.

The isopycnic technique, therefore, separates particles into zones solely on the basis of their buoyant density differences, independent of time. In many density gradient experiments, particles of both the rate zonal and the isopycnic principles may enter into the final separations. For example, the gradient may be of such a density range that one of the components sediments to its density in the tube and remains there, while another component sediments to the bottom of the tube. The self-generating gradient technique often requires long hours of centrifugation.

Isopynically banding DNA, for example, takes 36 to 48 hours in a self-generating cesium chloride gradient. It is important to note that the run time cannot be shortened by increasing the rotor speed; this only results in changing the position of the zones in the tube since the gradient material will redistribute further down the tube under greater centrifugal force.

Tags: Bio Technology, Bio Genetics , Biochemical Techniques

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