Biology Reference
In-Depth Information
15.
TPCK-Trypsin reagent: TPCK is added to trypsin to inactivate any residual
chymotrypsin activity, which would otherwise destroy the pyrrole. This reagent
should be freshly prepared on the day of use. Trypsin is dissolved in the TPCK
inactivator solution (1000 U/200
µ
L) and left at room temperature for 25 min to
inactivate any chymotrypsin.
16.
DAB reagent: 500 mg of 4-dimethylaminobenzaldehyde (DAB) is dissolved in
4.4 mL 60% perchloric acid and made up to 10 mL with water. Reagent blank is
prepared as above minus the DAB.
17.
Pyrrole standards: A standard curve is prepared using 1-methyl pyrrole. This is
obtainable as a liquid from Aldrich (Cat no. M7, 880-1). 11.1
µ
L is made up to
2.5 mL in ethanol from which 20
L is diluted to 100 mL in TAPSO/TPCK
reagent to give a final concentration of 10
µ
M
pyrrole. This stock solution is used
to produce a series of solutions in the concentration range 1-5
µ
µ
M
by dilution
according to the following table:
10
µ
M
Stock pyrrole,
µ
L
20
40
60
80
100
TAPSO/TPCK buffer,
µ
L
180
160
140
120
100
Pyrrole conc.,
µ
mol/L
1
2
3
4
5
3. Methods (seeNote 1)
3.1. Borohydride Reduction of Sample
1.
The weighed sample is finely comminuted (
see
Note 2
) and evenly dispersed in a
volume of phosphate buffered saline (0.15
M
sodium chloride, 0.05
M
sodium
phosphate pH 7.4) equal to between 5 and 10 times the volume of the sample (
see
Note 3
).
2.
A weight of sodium borohydride equal to 1% of the sample wet weight is dis-
solved in 0.001
M
sodium hydroxide at 4
°
C and this is added to the sample (
see
Note 4
).
3.
The temperature of the reduction mixture is raised to approximately 20
C and
reduction allowed to proceed for 1 h in a fume hood with occasional stirring.
After this period the mixture is acidified to approximately pH 3.0 by addition of
glacial acetic acid (pH paper accuracy is sufficient). (
See
Note 5.
)
°
4.
The acidified reducing reagents are now discarded either by filtration or after
centrifugation, however, it can often be simply achieved by carefully decanting
the reagents. The sample is then washed three times with distilled water in order
to remove both the acetic acid and salts from the reduction mixture prior to freeze-
drying (
see
Note 6
).
3.2. Hydrolysis of the Sample
1.
The weighed, dry sample is placed in a suitable vessel and hydrolysed in a vol-
ume of constant boiling hydrochloric acid to give a concentration of approx 5 mg
of sample / mL of acid (
see
Notes 7
and
8
).
2.
Seal the hydrolysis vessel and heat to 110
°
C for 24 h (
see
Note 9
).
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