BioFiles Volume 6, Number 5 — Centrifugation
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Table of Contents
Separation of particles by sedimentation is one of the most powerful tools in biology. Even though sedimentation using centrifugation is not a new technology, it is essential for cutting edge genomic and proteomic research by providing purified particles of interest. In a survey at the US National Institutes of Health, over 65% of research workers replied that using centrifugation to purify cells, subcellular organelles, viruses, proteins, and nucleic acids is an integral part of their work.1
Density gradient centrifugation is a technique that allows the separation of particles on the basis of their size, shape, and density. A density gradient is typically created by layering media of increasing density in a centrifuge tube. When a sample is layered on top of a density gradient and centrifuged, the various particles move through the gradient at different rates. The particles appear as bands or zones in the gradient with the more dense and larger particles migrating furthest.
A number of different compounds have been investigated as density gradient media. One of the first density gradient centrifugation techniques was developed in the 1950s and used a buffered sucrose solution for the purification of cell organelles. Sucrose quickly became the density medium of choice for separating homogenized mammalian tissues. Later, cesium chloride gradients were used to separate DNA of different densities. Meselson and Stahl in 1958 used cesium chloride density gradient centrifugation in an elegant experiment to support the semi-conservative model of DNA replication. Colloidal-silica suspensions were first manufactured by DuPont and sold under the name of LUDOX®.2
In 1977, the stabilized silica colloid coated with polyvinylpyrrolidone (PVP) called Percoll® became available for separating cells and subcellular particles. In 1968, Boyum described methods for the isolation of mononuclear cells from circulating blood and bone marrow using mixtures of polysaccharide and a radiopaque contrast medium. This led to the development of the first nonionic iodinated density gradient medium, metrizamide, in the 1970s.3 Now, a large selection of commercial iodinated density gradient media are available.
This issue of Biofiles examines the theory and practice of biological separation. The beginning section provides a primer on the basic concepts of centrifugation. Three basic types of centrifugal separations are highlighted; differential centrifugation, rate-zonal centrifugation, and isopycnic centrifugation. A concise description of each is given along with the separation principles involved. Cell viability kits, technical support information, and centrifugation equipment are also included.
We hope you find the reference information and products relevant. For a comprehensive list of our centrifugation media products, please visit sigma.com/handh.