Centrifugation Techniques

Abstract Centrifugation is the use of the centrifugal forces generated in a spinning rotor to separate biological particles, such as cells, viruses, sub‐cellular organelles, macromolecules (principally proteins and nucleic acids) and macromolecular complexes (such as ribonucleoproteins and lipoproteins). The three main methods of separation are differential pelleting, rate‐zonal centrifugation and isopycnic centrifugation. The first two methods separate particles primarily on the basis of size while isopycnic centrifugation separates particles on the basis of their density. The choice of centrifugation method depends on the nature of the particles and often more than one separation technique is required. For example, membrane fractionation often involves first making an enriched fraction from a cell homogenate by differential pelleting followed by isopycnic centrifugation to obtain purified fractions. Key Concepts Modern centrifuges and their rotors can generate centrifugal forces up to a million times that of the Earth's gravity that can separate very small particles. Depending on the centrifugation force used, particles from cells to those as small as macromolecules can be separated. Particles can be separated on the basis of their size using differential pelleting or rate‐zonal centrifugation. Alternatively, particles can be separated on the basis of their density using isopycnic centrifugation. Once cells are broken open by homogenisation, they can be fractionated into a range of defined membrane fractions so that it is possible to analyse the functions of the different components of cells. Many centrifugation purification procedures involve sequential centrifugation steps, often a size separation followed by purification using isopycnic gradient centrifugation.