Ultracentrifugation

Abstract

Centrifuges are used in cell and molecular biology departments, in industrial laboratory settings and medical laboratories. They are the workhorses for separating the components of heterogeneous mixtures or for purifying biological particles. Fundamental to centrifuges is the increase in the effective force of gravity = relative centrifugal force (rcf) ⇒ expressed in multiples of Earth's gravity (1g). This is accomplished by spinning the sample to be disaggregated in the single cavity or the tubes of a rotor as the ‘mobile part’ of a centrifuge. Subjected to the rcf thus applied, the constituents of a sample will differentially sediment according to their physical properties, like size and molecular mass as well as density and viscosity of the sample solution.

Modern ultracentrifuges accelerate an appropriate rotor to speeds up to >50 000 revolutions per minute (rpm), generating thereby an rcf up to the millionfold of the Earth's gravity. Efficiency and range of activities of these latest machines reflect the technological progress during the past 3–4 decades with the upcoming microchip technology, the construction of new heavy‐duty drive systems, the installation of a sophisticated control equipment and last but not least the access to the novel carbon‐fibre material in the design of rotors. While the classic floor‐standing ultracentrifuge has been revolutionised by these groundbreaking changes to its design, with the benchtop version, a novel model matching with the demands on flexibility and versatility of experimental working has been launched in the meantime.

Basically, there are two kinds of ultracentrifuges, preparative and analytical ones. The former is widely used to fractionate tissue homogenates or cell suspensions, aiming to isolate and purify the distinct classes of biological material: intact cells, subcellular organelles, macromolecules and corresponding complexes, bacteria or viruses. By means of analytical centrifuges, the physico‐chemical properties of a sedimenting particle or molecular interactions of the subunits of multiprotein complexes can be unravelled.

Key Concepts

  • Centrifugation is widely used in studying fundamental cellular processes such as the inter‐ and intracellular transport of metabolites, the biosynthesis and breakdown of proteins or the molecular interactions of the components of multiprotein complexes.
  • Ultracentrifugation is basically carried out in two ways: preparative and analytical centrifugation.
  • The former aims to isolate and purify subcellular organelles or multiprotein complexes; the latter allows to analyse the mutual interactions between the subunits of multiprotein complexes and to unravel physico‐chemical properties like the mass and size of macromolecules.
  • Preparative centrifugation may be performed as batch‐type (conventional) centrifugation or alternatively in the continuous‐flow mode.
  • The former is mostly used to separate and enrich organelles out of complex biological mixtures commonly limited in their quantity.
  • Continuous‐flow centrifugation is particularly useful for the large‐scale collection of particles out of diluted solutions or suspensions (bacteria, viruses) as it combines high centrifugal forces with high throughput.
  • Theoretically, two kinds of preparative centrifugation have to be distinguished: differential centrifugation and density gradient centrifugation.
  • Differential centrifugation fractionates organelles according to their size, mass and shape yet leads only to an enriched rather than a highly purified preparation of a particular organelle.
  • To get such a preparation genuinely purified, contaminants have to be removed by density gradient centrifugation.
  • Rate zonal and isopycnic density gradient centrifugation differ in their basic concepts and the types of density gradients employed.

Keywords: cell fractionation; ultracentrifuge; rotor; density gradient; preparative and analytical centrifugation; conventional and continuous‐flow centrifugation

Figure 1. Differential pelleting.
Figure 2. Flow chart summarising the sequential steps in the subfractionation of a homogenate by differential centrifugation.
Figure 3. The density of a gradient may be increased continuously or discontinuously from top to bottom of the centrifuge tube.
Figure 4. Modes of density gradient centrifugation: (upper) rate zonal centrifugation and (lower) isopycnic centrifugation.
Figure 5. Photograph of an actual tissue separation: subfractionation of a rat hepatic light mitochondrial fraction by rate zonal centrifugation using a vertical tube rotor.
Figure 6. Buoyancy (B), friction (F) and centrifugal (C) forces acting on a particle during centrifugation.
Figure 7. A vertical tube rotor.
Figure 8. A fixed‐angle rotor.
Figure 9. A swing‐out rotor.
close

References

Anderson NG (1966) Zonal centrifuges and other separation systems. Science (New York) 154: 103–112.

Birnie GD (ed) (1972) Subcellular Components: Preparation and Fractionation, 2nd edn. London: Butterworths.

Claude A (1946) Fractionation of mammalian liver cells by differential centrifugation. 1: problems, methods, and preparation of extract. Journal of Experimental Medicine 84: 51–59.

De Duve C (1971) Tissue fractionation. Past and present. Journal of Cell Biology 50: 20D–55D.

De Duve C (1975) Exploring cells with a centrifuge. Science (Washington, DC) 189: 186–194.

Drahos KL, Tran HC, Kiri AN, et al. (2005) Comparison of Golgi apparatus and endoplasmic reticulum proteins from livers of juvenile and aged rats using a novel technique for separation and enrichment of organelles. Journal of Biomolecular Techniques 16: 347–355.

Hinton RH, Mullock BM and Gilhuus‐Moe CC (1974) In: Reid E (ed) Methodological Developments in Biochemistry, vol. 4, p. 103. London: Longmans.

Kiri AN, Tran HC, Drahos KL, et al. (2005) Proteomic changes in bovine heart mitochondria with age: using a novel technique for separation and enrichment. Journal of Biomolecular Techniques 16: 371–379.

Miescher F (1871) Über die Zusammensetzung der Eiterzellen. Medizinisch‐chemische Untersuchungen 4: 441–460.

Sanchez‐Lopez V, Fernandez‐Romero JM and Gomez‐Hens A (2009) Evaluation of liposome populations using a sucrose density gradient centrifugation approach coupled to a continuous flow system. Analytica Chimica Acta 645: 79–85.

Schumaker VN (1967) Zone centrifugation. Advances in Biological and Medical Physics 11: 245–339.

Svedberg T and Pedersen KO (1940) The Ultracentrifuge. Oxford: Clarendon Press.

Further Reading

Birnie GD and Rickwood D (eds) (1978) Centrifugal Separations in Molecular and Cell Biology. London: Butterworths.

Dorin M and Cummings J (2004) Principles of Continuous Flow Centrifugation. Technical Information, Beckman Coulter. www.Beckman.com/literature/Bioresearch/T‐1780B

Ford TC and Graham JM (eds) (1991) An introduction to Centrifugation. Oxford: BIOS Scientific Publishers Limited.

Price CA (ed.) (1982) Centrifugation in Density Gradients. New York: Academic Press.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Mohr, Heribert, and Völkl, Alfred(May 2017) Ultracentrifugation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002969.pub3]