Sucrose gradient centrifugation

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Sucrose gradient centrifugation
Sucrose gradient centrifugation is a type of centrifugation often used to purify enveloped viruses (with densities
1.1-1.2 g/cm), ribosomes, membranes and so on. This method is also used to purify exosomes.[1] There are two
methods - equilibrium centrifugation and non-equilibrium centrifugation.
Typically in equilibrium centrifugation, a sucrose density gradient is created by gently overlaying lower
concentrations of sucrose on higher concentrations in a centrifuge tube. For example, a sucrose gradient may consist
of layers extending from 70% sucrose to 20% sucrose in 10% increments (though this is highly variable depending
on sample to be purified).
Alternatively, devices known as gradient mixers or gradient makers can be used to form a gradient. These devices
consist of two chambers, containing solutions of differing concentrations, which are gradually mixed to create the
gradient. These devices can be used to create linear, concave, convex and exponential gradients.
The sample containing the particles of interest is placed on top of the gradient and centrifuged at forces in excess of
150,000 x g. The particles travel through the gradient until they reach the point in the gradient at which their density
matches that of the surrounding sucrose. This fraction can then be removed and analyzed. After it becomes known
between which two layers the required fraction finally settles, a simplified setup with just these two layers may be
used.
A similar technique is sucrose cushion centrifugation, in which a particle mixture is pelleted through a 20% sucrose
layer, coming to rest at the interface with a 70% solution. This allows concentration of particles from a sample.
Unlike standard centrifugation, which in effect crushes the particles against the bottom of the centrifuge tube, the
sucrose cushion method causes no mechanical stress and allows the collection of morphologically intact
particles[citation needed].
Non-equilibrium centrifugation is very similar to the equilibrium form, but the experiment is only run until a
particular point. (These gradients can be called "velocity gradients"). Although particles with more density/less drag
travel farther from the top surface, the run is ended before equilibrium is reached. The desired particle will be at a
(hopefully known) set distance from the surface, and that band of the gradient is collected (sometimes called a
"fraction").
Collection, once the run is ended, can be done by several methods. Perhaps the easiest way is for the sucrose
solution to be eluted (drained)by puncturing the bottom of the tube and only the part with the desired protein or
material is kept. It is also possible to suction the sucrose (gently, so as not to mix the layering formed during the
centrifugation), dividing the suctioned material into successive "fractions". These can then be assayed by any of a
number of means, to determine the distribution of the desired material, which allows you to choose the fractions
containing this molecule/material.