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Methods Mol Biol. 2011;777:29-55. doi: 10.1007/978-1-61779-252-6_3.

Purification of tubulin from the fission yeast Schizosaccharomyces pombe.

Author information

1
Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK. d.drummond@mechanochemistry.org

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.

PMID:
21773919
DOI:
10.1007/978-1-61779-252-6_3
[Indexed for MEDLINE]

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