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3 - Centrifugation

Keith Wilson
Affiliation:
University of Hertfordshire
John Walker
Affiliation:
University of Hertfordshire
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Summary

INTRODUCTION

Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and isolated macromolecules such as proteins or nucleic acids. The development of the first analytical ultracentrifuge by Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades. Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all invasive subcellular studies. While analytical centrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies, preparative centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest.

Most undergraduate students will be exposed to preparative centrifugation protocols during practical classes and might also experience a demonstration of analytical centrifugation techniques. This chapter is accordingly divided into a short introduction into the theoretical background of sedimentation, an overview of practical aspects of using centrifuges in the biochemical laboratory, an outline of preparative centrifugation and a description of the usefulness of ultracentrifugation techniques in the biochemical characterisation of macromolecules. To aid in the understanding of the basic principles of centrifugation, the general design of various rotors and separation processes is diagrammatically represented.

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Publisher: Cambridge University Press
Print publication year: 2010

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References

Burgess, N. K., Stanley, A. M. and Fleming, K. G. (2008). Determination of membrane protein molecular weights and association equilibrium constants using sedimentation equilibrium and sedimentation velocity. Methods in Cell Biology, 84, 181–211. (Focuses on the centrifugal analysis of interactions between integral membrane proteins.)CrossRefGoogle ScholarPubMed
Cole, J. L., Lary, J. W., Moody, T. P. and Laue, T. M. (2008). Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium. Methods in Cell Biology, 84, 143–179. (Provides an excellent synopsis of the applicability of ultracentrifugation to the characterisation of macromolecular behaviour in complex solution.)CrossRefGoogle ScholarPubMed
Cox, B. and Emili, A. (2006). Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Nature Protocols, 1, 1872–1878. (Outlines differential centrifugation protocols for the isolation of the nuclear, cytosolic, mitochondrial and microsomal fraction.)CrossRefGoogle ScholarPubMed
Girard, M., Allaire, P. D., Blondeau, F. and McPherson, P. S (2005). Isolation of clathrin-coated vesicles by differential and density gradient centrifugation. Current Protocols in Cell Biology, Chapter 3, Unit 3.13. (Describes a typical subcellular fractionation protocol used in modern biochemical applications.)CrossRef
Klassen, R., Fricke, J., Pfeiffer, A. and Meinhardt, F. (2008). A modified DNA isolation protocol for obtaining pure RT-PCR grade RNA. Biotechnology Letters, 30, 1041–1044. (Describes typical centrifugation protocol used for the isolation of DNA and RNA molecules.)CrossRefGoogle ScholarPubMed

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  • Centrifugation
  • Edited by Keith Wilson, University of Hertfordshire, John Walker, University of Hertfordshire
  • Book: Principles and Techniques of Biochemistry and Molecular Biology
  • Online publication: 05 July 2013
  • Chapter DOI: https://doi.org/10.1017/CBO9780511841477.004
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  • Centrifugation
  • Edited by Keith Wilson, University of Hertfordshire, John Walker, University of Hertfordshire
  • Book: Principles and Techniques of Biochemistry and Molecular Biology
  • Online publication: 05 July 2013
  • Chapter DOI: https://doi.org/10.1017/CBO9780511841477.004
Available formats
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Save book to Google Drive

To save content items to your account, please confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your account. Find out more about saving content to Google Drive.

  • Centrifugation
  • Edited by Keith Wilson, University of Hertfordshire, John Walker, University of Hertfordshire
  • Book: Principles and Techniques of Biochemistry and Molecular Biology
  • Online publication: 05 July 2013
  • Chapter DOI: https://doi.org/10.1017/CBO9780511841477.004
Available formats
×