Density Gradient Centrifugation
Density gradient centrifugation is a technique that allows the separation of cells, organelles and macromolecules, depending on their size, shape and density.
A density gradient is created in a centrifuge tube by layering solutions of varying densities with the dense end at the bottom of the tube. Cells and large molecules are usually separated on a shallow gradient of sucrose or other inert carbohydrates even at relatively low centrifugation speeds, while macromolecules such as proteins and nucleic acids are separated at higher centrifugation using ultracentrifuges.
When mixtures of cellular extracts are layered on top of a density gradient in a tube and subjected to centrifugation, the various components move through the gradient at different rates that are dependant on their sizes and shapes. These different components appear as distinct bands or zones in the gradient with large components migrating farthest in the tube in a given period of time. The rate with which a fraction moves the fixed distance in the gradient tube is dependant of its sedimentation value (S) that, in turn is determined by the size and shape of that fraction. By comparing the different position of the components in the gradient, it is possible to make an approximate measurement of their molecular weight. It is, however, difficult to precisely determine these molecular weights, as this requires knowledge about the shape of these molecules, which is hard to determine with accuracy. This density gradient separation technique is called rate zonal centrifugation and is usually performed with a shallow sucrose gradient. The different components being separated by this technique are denser than any of the sucrose concentrations used in the gradient. Samples are, therefore, centrifuged just long enough to separate the components of interest. Longer centrifugation than necessary would allow all components to form a pellet at the bottom of the tube. One of the most important applications of this technique over the past decades was the separation of transfer RNA (4S) from ribosomal RNA that forms three different classes with distinct sedimentation values 23S, 16S and 5S. This helped to facilitate the characterization of the protein synthesizing system.
A second density gradient technique, called equilibrium density-gradient centrifugation is used to separate cellular components on the basis of their buoyant density. In this case the cellular mixture is centrifuged through a steep density gradient that contains a high concentration of sucrose, or more often, cesium chloride (CsCl). In these gradients, the molecules being studied have a density somewhere in between the highest and lowest densities of sucrose or CsCl generated in the gradient. The components of a sample begin to move down this gradient in the same way as they do in a rate-zonal density gradient. When a component of the mixture reaches a point where the density of the solution is equal to its own density, it stops moving further and forms a distinct band. The position of the band in the tube is characteristic of the buoyancy of that component. Buoyancy, or buoyant density of a substance is its tendency to float in a medium, which in this case is the density gradient.
Equilibrium density gradient centrifugation using CsCl was for decades the method of choice in the purification of highly pure plasmid DNA. Meselson and Stahl, who developed this technique, were the first to use it in an experiment that provided evidence for the semi-conservative replication of DNA and confirmed the double helix structure of DNA proposed by Crick and Watson.
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