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.

Keywords: differential pelleting; isopycnic; rate‐zonal; sucrose; CsCl ; Nycodenz; iodixanol (commercially available as OptiPrep™) ; membranes; viruses; cells

Figure 1. Flow chart of a typical differential centrifugation of a cell or tissue homogenate. Principal components of fractions in italics.
Figure 2. Differential centrifugation of a homogenate: (a) before centrifugation and (b) after centrifugation.
Figure 3. Rate‐zonal or sedimentation velocity density gradient centrifugation. (a) Sample is loaded in a narrow band on top of a continuous density gradient. (b–d) With increasing centrifugation time, the particles in the sample zone move down through the gradient as discrete bands.
Figure 4. Buoyant density gradient centrifugation in a continuous gradient. The figure describes the separation of three types of particle: red, green and magenta (density increasing in that order). The sample may be layered on top (1), beneath (2) or be made part of (3) the gradient (see text for details).
Figure 5. Banding of lymphocytes from human blood on a density barrier (Lymphoprep™); before (left) and after centrifugation (right).
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References

Dettenhofer M and Yu XF (1999) Highly purified human immunodeficiency virus type 1 reveals a virtual absence of V if in virions. Journal of Virology 73: 1460–1467.

Dobrota M and Hinton R (1992) In: Rickwood D , (ed). Preparative Centrifugation – a Practical Approach, pp. 77–142. Oxford: IRL Press at Oxford University Press.

Graham J , Ford T and Rickwood D (1994) The preparation of subcellular organelles from mouse liver in self‐generated gradients of iodixanol. Analytical Biochemistry 220: 367–373.

Hermens WTJMC , Ter Brake O , Dijkhuizen PA , et al. (1999) Purification of recombinant adeno‐associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system. Human Gene Therapy 10: 1885–1891.

McCaffrey G , Welker J , Scott J , van der Salm L and Grimes ML (2009) High‐resolution fractionation of signaling endosomes containing different receptors. Traffic 10: 938–950.

Pryor S , McCaffrey G , Young LR and Grimes ML (2012) NGF causes TrkA to specifically attract microtubules to lipid rafts. PLoS One 7: e35163.

Zolotukhin S , Byrne BJ , Mason E , et al. (1999) Recombinant adeno‐associated virus purification using novel methods improves infectious titer and yield. Gene Therapy 6: 973–985.

Further reading

Preparative Centrifugation: A Practical Approach (1992) Edited D Rickwood , Oxford: Oxford University Press.

Subcellular Fractionation: A Practical Approach (1997) Edited J Graham and D Rickwood , Oxford University Press: Oxford.

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How to Cite close
Rickwood, David, and Graham, John(Apr 2015) Centrifugation Techniques. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002704.pub2]