Biology Reference
In-Depth Information
methanol such that the final stock has 50:50 methanol/buffer. The advantage of
adding methanol is that the mixture remains liquid at
20 C thus facilitating
subsequent handling. Aliquot calibrationmixture and store at
80 Cuntil analysis.
￿ When working with extremely thermo-instable compounds, such as redox
cofactors, exempt them from the calibration mixture. Rather, prepare fresh
standards directly before analysis.
For calibration, a constant amount of U- 13 C internal standard is mixed with a dilution
series of the calibration mixture. The range of concentrations in the dilution series
should ideally cover the whole range of concentrations that occurs in the cell extract.
￿ Prepare seven levels of metabolite standard by a 1:3 dilution series of the
metabolite calibration mixture. The resulting concentrations are 100, 33.3, 11.1,
3.7, 1.23, 0.41 and 0.14
m
M.
￿ Mix 15
m
L of each metabolite standard with 15
m
L of the internal standard.
￿ Vacuum dry the standards and re-suspend in 15
m
L pure water.
; i of the signal of 12 C metabolites and the signal of the
corresponding U- 13 C-labelled metabolites. This ratio equals the moles of 12 C
metabolite in the calibration level j
￿ Calculate the ratio
n cal j
i
and U- 13 C metabolite from the internal
ð
Þ
standard ð
n IS
i
Þ .
c cal i V cal
c I i V IS
where c cal i and V cal are the concentration and the volume of the calibration level j
added (here 15
n cal j
i
; i ¼
n IS i ¼
L) and V IS
m
is the volume of the internal standard added (here
L). The unknown concentration in the internal standard c IS
i
15
m
follows from:
c cal i V cal
c IS
i
i
; i V IS
¼
i
Since the ratio is determined at different calibration levels c cal i , use a regression
analysis to get the best estimate for c I i . Usually, a linear regression of ratio versus
concentration in the calibration level will work.
￿ Using the thereby estimated concentration of the internal standard c I i , the number
of moles of metabolite i extracted from bacteria follows from:
n cell
i
¼; i n IS i ¼; i c I i V IS
5.5 Normalizing quantified metabolites to biomass or total
cell volume
In order to obtain quantitative data, the ratio
; i or the extracted moles n i , which have
been determined as described in Section 5.4, have to be normalized. Usually, the
amount of extracted biomass m x is given as mL OD or mg DryWeight . For comparison
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