7.2.3. Gradient Centrifugation
The purpose of gradient centrifugation is to concentrate the virus particles according to their specific density and thereby separate them from other cellular material with different density. For high purity requirements, where the virus needs to be separated from other particles with similar density (e.g. ribosomes), ‘continuous’ gradients are used. These have a gradual transition from high to low density so that each particle-type can band at its own specific density. For lower purity requirements ‘discontinuous’ gradients can be used. These have low density solution layered on top of high density solution, with a sharp interface between them where all material with a specific density between the high and low solutions concentrates. Discontinuous gradients are slightly easier to prepare and to fractionate, but continuous gradients are cleaner and more secure if the specific density of a virus is not known. Many different substances can be used for creating the density differential (sugars, salts, polyethylene glycol, synthetic polymers), each with their (dis)advantages, but for most virus purification purposes sucrose gradients are adequate. The most common alternative is CsCl (caesium chloride) gradients. These are easier to prepare and generally leave cleaner virus preparations, but require longer centrifugation. CsCl is also chaotropic, stripping virus particles from other cellular constituents, and is therefore not suitable for purifying enveloped viruses. For these, sucrose gradients should be used. Gradient centrifugation is best done in a high-speed, swing-out rotor. These usually have 6 buckets, which should all be used during each centrifugation run, since the rotor is only fully balanced when each bucket is present in its correct position. This means that for each run, six centrifugation tubes should be prepared and balanced.