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Home » Medical Terminology » Density Gradient Ultracentrifugation      

Density Gradient Ultracentrifugation

Ultracentrifugation methods sorting macromolecules by sedimentation rates have been widely used for biochemical and pharmaceutical purposes. Many brilliant achievements in molecular biology were accomplished with the ultracentrifuge, including the mechanism of DNA replication and the discovery of ribosomes. 6 Determination of high-density cholesterol and HIV-1 virus in plasma are also good examples.

Density gradient ultracentrifugation (DGUC) is a common separation technique based on size and density of analyzed particles. It is a high-speed centrifugation step in which a solution of low molecular weight, for example, cesium chloride (CsCl) is centrifuged in a cell, so that a density gradient is formed at equilibrium. If a substance of high molecular weight is added to this solution then it will be suspended at a position where its buoyant density is equal to the density of the low molecular weight solution. The position where it is suspended is measured and is used to calculate its molecular weight. 5 The usefulness of DGUC is threefold: it provides information on the size of the separated molecules in a mixture, permits separation on a preparative scale, and can be carried out with low concentrations of solute. 3

Density gradient ultracentrifugation has been widely used to fractionate animal, plant and bacterial cells, viral particles, lysosomes, membranes, and macromolecules in a variety of processes. Its application has been very important in the commercial preparation of viruses for vaccine and immunotherapy products such as influenza, hepatitis, and rabies vaccines. 2

Cesium chloride (CsCl) DGUC was routinely used in the past to isolate DNA. The densities of DNA are about the same as those of concentrated solutions of cesium chloride. Centrifugation of CsCl solutions at very high rotational speeds, where the centrifugal force becomes 105 times stronger than the force of gravity, causes the formation of a density gradient within the solution. This gradient is the result of a balance that is established between the sedimentation of the salt ions toward the bottom of the tube and their diffusion toward regions of lower concentration. If DNA is present in centrifuged CsCl solution, it moves to a position of equilibrium in the gradient equivalent to its buoyant density. 4

Density gradient ultracentrifugation methods have been used to separate the major lipoprotein classes, including HDL, in a single spin. The major HDL subclasses (HDL2, density range 1.063-1.125 kg/L, and HDL3, density range 1.125-1.210 kg/L) and even some minor subclasses can also be separated under certain conditions. 1

References

  1. Nader Rifai, G. Russell Warnick, Marek H. Dominiczak. Handbook of Lipoprotein Testing
  2. Mohamed A. Desai. Downstream Processing of Proteins
  3. Yeshajahu Pomeranz, Clifton E. Meloan. Food Analysis: Theory and Practice
  4. Reginald Garrett, Charles M. Grisham. Biochemistry
  5. Vasantha Pattabhi, N. Gautham. Biophysics
  6. Igor N. Serdyuk, Nathan R. Zaccai, Giuseppe Zaccai. Methods in Molecular Biophysics: Structure, Dynamics, Function


 










 





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