Purification of tubulin from the fission yeast schizosaccharomyces pombe

Douglas R. Drummond, Susan Kain, Anthony Newcombe, Christina Hoey, Miho Katsuki, Robert A. Cross

研究成果: Chapter in Book/Report/Conference proceedingChapter

21 被引用数 (Scopus)

抄録

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.

本文言語英語
ホスト出版物のタイトルMicrotubule Dynamics
ホスト出版物のサブタイトルMethods and Protocols
編集者Anne Straube
ページ29-55
ページ数27
DOI
出版ステータス出版済み - 2011
外部発表はい

出版物シリーズ

名前Methods in Molecular Biology
777
ISSN(印刷版)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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