Purification of tubulin from the fission yeast schizosaccharomyces pombe

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

研究成果: 著書/レポートタイプへの貢献

18 引用 (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.

元の言語英語
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
ページ29-55
ページ数27
DOI
出版物ステータス出版済み - 8 3 2011
外部発表Yes

出版物シリーズ

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

Fingerprint

Schizosaccharomyces
Tubulin
Chromatography
Anion Exchange Resins
Ammonium Sulfate
Microtubules
Polymerization
Gel Chromatography
Anions
Protein Isoforms
Proteins
Nitrogen
Salts

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

これを引用

Drummond, D. R., Kain, S., Newcombe, A., Hoey, C., Katsuki, M., & Cross, R. A. (2011). Purification of tubulin from the fission yeast schizosaccharomyces pombe. : Microtubule Dynamics: Methods and Protocols (pp. 29-55). (Methods in Molecular Biology; 巻数 777). https://doi.org/10.1007/978-1-61779-252-6_3

Purification of tubulin from the fission yeast schizosaccharomyces pombe. / Drummond, Douglas Robert; Kain, Susan; Newcombe, Anthony; Hoey, Christina; Katsuki, Miho; Cross, Robert A.

Microtubule Dynamics: Methods and Protocols. 2011. p. 29-55 (Methods in Molecular Biology; 巻 777).

研究成果: 著書/レポートタイプへの貢献

Drummond, DR, Kain, S, Newcombe, A, Hoey, C, Katsuki, M & Cross, RA 2011, Purification of tubulin from the fission yeast schizosaccharomyces pombe. : Microtubule Dynamics: Methods and Protocols. Methods in Molecular Biology, 巻. 777, pp. 29-55. https://doi.org/10.1007/978-1-61779-252-6_3
Drummond DR, Kain S, Newcombe A, Hoey C, Katsuki M, Cross RA. Purification of tubulin from the fission yeast schizosaccharomyces pombe. : Microtubule Dynamics: Methods and Protocols. 2011. p. 29-55. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-61779-252-6_3
Drummond, Douglas Robert ; Kain, Susan ; Newcombe, Anthony ; Hoey, Christina ; Katsuki, Miho ; Cross, Robert A. / Purification of tubulin from the fission yeast schizosaccharomyces pombe. Microtubule Dynamics: Methods and Protocols. 2011. pp. 29-55 (Methods in Molecular Biology).
@inbook{21e950372e65450ba6d97fb53636cc3b,
title = "Purification of tubulin from the fission yeast schizosaccharomyces pombe",
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.",
author = "Drummond, {Douglas Robert} and Susan Kain and Anthony Newcombe and Christina Hoey and Miho Katsuki and Cross, {Robert A.}",
year = "2011",
month = "8",
day = "3",
doi = "10.1007/978-1-61779-252-6_3",
language = "English",
isbn = "9781617792519",
series = "Methods in Molecular Biology",
pages = "29--55",
booktitle = "Microtubule Dynamics",

}

TY - CHAP

T1 - Purification of tubulin from the fission yeast schizosaccharomyces pombe

AU - Drummond, Douglas Robert

AU - Kain, Susan

AU - Newcombe, Anthony

AU - Hoey, Christina

AU - Katsuki, Miho

AU - Cross, Robert A.

PY - 2011/8/3

Y1 - 2011/8/3

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=79960916482&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79960916482&partnerID=8YFLogxK

U2 - 10.1007/978-1-61779-252-6_3

DO - 10.1007/978-1-61779-252-6_3

M3 - Chapter

SN - 9781617792519

T3 - Methods in Molecular Biology

SP - 29

EP - 55

BT - Microtubule Dynamics

ER -