Biology Reference
In-Depth Information
￿ Dry the supernatants at 0.12 mbar to complete dryness in a vacuum concentrator.
Dry metabolite extracts can be stored at
80 C until analysis.
￿ Before analysis re-suspend dry metabolite extracts in 100
m
L water.
g for 10 min at 4 C and use 10-20
￿ Centrifuge at 12,000
m
L of the supernatant
for mass spectrometry analysis.
4.2 Hot ethanol extraction of whole cell broth
￿ Prepare 4 mL 75:25 ethanol (p.a.)/water in 15 mL reaction tubes and preheat to
78 C in water bath (will yield a 60:40 ethanol:water ratio after addition of
sample).
￿ Transfer 1 mL whole culture broth into extraction solution and mix by vigorous
shaking for 3 s.
￿ Add internal standard (e.g. U- 13 C-labelled metabolite extract).
￿ Incubate 3 min at 78 C. If you observe sedimentation of cells, shake vigorously
every minute.
￿ Separate cell debris and metabolite extract by centrifugation at 12,000
g for
10 min at 4 C.
￿ Transfer supernatant to a fresh 15 mL tube.
￿ Add 1 mL 75:25 ethanol (p.a.)/water at 78 C to cell pellet, re-suspend and
incubate at 78 C for 1 min.
￿ Separate cell debris and metabolite solution by centrifugation at 12,000
g for
4 C.
￿ Add this supernatant to the supernatant from the first extraction and dry at
0.12 mbar to complete dryness in a vacuum concentrator. Dry metabolite extracts
can be stored at
10 min at
80 C until analysis.
￿ Before analysis, re-suspend dry metabolite extracts in 100
L water. Depending
on the medium composition, the sample might not dissolve completely.
￿ Centrifuge at 12,000
m
g for 10 min at 4 C and use 10-20 m L of the supernatant
for mass spectrometry analysis.
￿ Carefully transfer 10
L of clear supernatant to a vial or well plate used for
analysis and dilute with 40-90
m
m
L water (or mobile phase at start of LC gradient)
to reduce salt concentration.
4.3 Acidic acetonitrile extraction of filtered samples
This method, adopted from that of the Rabinowitz group, is used to quantify nucle-
otide triphosphates that decompose during hot ethanol extraction ( Kimball and
Rabinowitz, 2006; Bennett et al. , 2008 ).
￿ Prepare 4 mL of 40:40:20 acetonitrile/methanol/water with 0.1 M formic acid in
small glass bottles and cool them to
20 C in a freezer.
￿ Transfer a nitrocellulose filter disc with the sample into the extraction solution
(see Section 2 ). Ensure the filter is completely immersed in the cold acetonitrile
solution.
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