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.
| Original language | English |
|---|---|
| Title of host publication | Microtubule Dynamics |
| Subtitle of host publication | Methods and Protocols |
| Editors | Anne Straube |
| Pages | 29-55 |
| Number of pages | 27 |
| DOIs | |
| Publication status | Published - Aug 3 2011 |
| Externally published | Yes |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 777 |
| ISSN (Print) | 1064-3745 |
Fingerprint
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Genetics
Cite this
Purification of tubulin from the fission yeast schizosaccharomyces pombe. / Drummond, Douglas R.; Kain, Susan; Newcombe, Anthony; Hoey, Christina; Katsuki, Miho; Cross, Robert A.
Microtubule Dynamics: Methods and Protocols. ed. / Anne Straube. 2011. p. 29-55 (Methods in Molecular Biology; Vol. 777).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Purification of tubulin from the fission yeast schizosaccharomyces pombe
AU - Drummond, Douglas R.
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
C2 - 21773919
AN - SCOPUS:79960916482
SN - 9781617792519
T3 - Methods in Molecular Biology
SP - 29
EP - 55
BT - Microtubule Dynamics
A2 - Straube, Anne
ER -