Yeast Functional Analysis Report
A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol
Article first published online: 28 APR 1999
Copyright © 1997 John Wiley & Sons, Ltd.
Volume 13, Issue 14, pages 1347–1355, November 1997
How to Cite
Gonzalez, B., François, J. and Renaud, M. (1997), A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol. Yeast, 13: 1347–1355. doi: 10.1002/(SICI)1097-0061(199711)13:14<1347::AID-YEA176>3.0.CO;2-O
- Issue published online: 28 APR 1999
- Article first published online: 28 APR 1999
- Manuscript Accepted: 16 APR 1997
- Manuscript Received: 18 FEB 1997
- French Ministry of Education
- yeast metabolism;
- metabolite extraction;
- metabolic engineering
A simple and reliable method for the efficient inactivation of metabolism and for quantitative metabolite extraction from yeast cells is presented. It is based on the use of a boiling solution made of 75% ethanol (volume/final volume) buffered with 70 mm-Hepes (final concentration), pH 7·5, to guarantee the stability throughout the whole procedure of a large variety of metabolites, including all glycolytic intermediates, nucleotides, pyridine nucleotides and organic acids compounds. The extraction is fast, requiring only 3 min incubation of yeast cells in the ethanol-buffered mixture maintained at 80°C. It can be carried out either directly by spraying the cells into the boiling mixture, or after quenching the whole culture in 60% methanol kept at −40°C. Extracts are subsequently concentrated by evaporation under partial vacuum and the residue is resuspended in a small volume of water. This concentration step and the use of a highly sensitive analytical method allow us to quantify metabolites in less than 10 mg dry weight cells. This method, which can be applied to other fungi, could be very helpful for the determination of true metabolites in mutants generated through the EUROFAN programme and for metabolic flux analysis. © 1997 John Wiley & Sons, Ltd.