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