A rapid and reliable method for metabolite extraction in yeast using boiling buffered ethanol

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

“…For measurement of metabolites in total broth as well as in the culture filtrate the same quenching procedure was applied, however, the quenched total broth mixtures (B) or quenched culture filtrates (F) were not centrifuged but after thorough vortexing 500 μL of these mixtures were withdrawn for metabolite extraction. Extraction of metabolites from the cell pellets as well as from the 500 μL samples from the quenched total broth was performed with the hot ethanol method (Gonzalez et al, 1997). In order to analyze the metabolites in the supernatants obtained from the quenching and washing procedure (QS and WS) and quenched culture filtrate (F), 500 μL of each of these solutions were subjected to the same procedure as for cell pellets.…”

Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase

“…Recoveries after subtracting cellular metabolite contributions ranged from 43-118% and were consistent for each metabolite. We also tested other cell harvesting procedures including transfer to cold buffered methanol as reported by others [19,20], but we found the most important variable in terms of quenching to prevent changes in levels of metabolites of interest was the addition of cycloheximide and sodium azide prior to harvesting cells. Other extraction procedures, e.g.…”

“…The pellets were washed twice with water, then dried for [16][17][18][19][20] h in open tubes placed in a block heater set at 60°C. Dry weights were measured after cooling the tubes for 1-2 h at room temperature; we found that a constant weight was reached using this procedure.…”

Global metabolite analysis of yeast: evaluation of sample preparation methods

“…Gonzalez et al (1997) found significant amounts of glucose 6-phosphate, ATP and NAD + in the supernatant from methanol-quenched cells. However, the amount of these metabolites was roughly equal to those found in the culture medium.…”

“…Another popular extraction method for intracellular metabolites of yeast is based on the use of boiling ethanol as first described by Entian et al (1977) and later revised by Gonzalez et al (1997). It is widely used for the extraction of intracellular metabolites of microorganisms (Tweeddale et al, 1998;Hans et al, 2001;Lange et al, 2001;Visser et al, 2002;Castrillo et al, 2003).…”