Sucrose gradient centrifugation

Jump to: navigation, search


Overview

Sucrose gradient centrifugation is a type of centrifugation often used to purify enveloped viruses (with densities 1.1-1.2 g/cm³) and ribosomes, and also to separate cell organelles from crude cellular extracts. This method is also used to purify exosomes.[1]

Equilibrium centrifugation

Typically, 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). 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 subjected to further analysis.

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.

Non-equilibrium centrifugation

This is very similar to the equilibrium form, but the experiment is only run until a particular point. Then the sucrose is eluted from the bottom of the tube.

References

  1. {{cite journal | author=Raposo, G., Nijman, H. W., Stoorvogel, W. et al.| title=B lymphocytes secrete antigen-presenting vesicles.| journal=J. Exp. Med. | year=1996 | volume=183| pages=1161 | url=http://www.jem.org/cgi/content/abstract/183/3/1161?ijkey=92acd76957442efd160b99c26777ac331fc23cc8&keytype2=tf_ipsecsha
de:Dichtegradientenzentrifugation



Linked-in.jpg