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Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe

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Microtubule Dynamics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 777))

Abstract

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies. S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a ­chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting step. The purified tubulin is concentrated, and then frozen and stored in liquid nitrogen.

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Acknowledgements

We gratefully acknowledge the Medical Research Council (G0200542), Association for International Cancer Research (09-0221), and Marie Curie Cancer Care for supporting this study.

Author information

Authors and Affiliations

  1. Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK

    Douglas R. Drummond, Miho Katsuki & Robert A. Cross

  2. Lister Hospital, Stevenage, UK

    Susan Kain

  3. Pfizer Biotechnology Ireland, Ringaskiddy, Ireland

    Anthony Newcombe

  4. Institute of Structural Molecular Biology, Birkbeck College, London, UK

    Christina Hoey

Authors
  1. Douglas R. Drummond
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  2. Susan Kain
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  3. Anthony Newcombe
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  4. Christina Hoey
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  5. Miho Katsuki
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  6. Robert A. Cross
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Corresponding author

Correspondence to Douglas R. Drummond .

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Editors and Affiliations

  1. Warwick Medical School, Ctr for Mechanochemical Cell Biology, University of Warwick, Coventry, CV4 7AL, United Kingdom

    Anne Straube

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Drummond, D.R., Kain, S., Newcombe, A., Hoey, C., Katsuki, M., Cross, R.A. (2011). Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe . In: Straube, A. (eds) Microtubule Dynamics. Methods in Molecular Biology, vol 777. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-252-6_3

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  • DOI: https://doi.org/10.1007/978-1-61779-252-6_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-251-9

  • Online ISBN: 978-1-61779-252-6

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