Biology Reference
In-Depth Information
3.5. CF-1 Prefractionation
1.
The dried sample is rehydrated in 0.5 mL water followed by 0.5 mL glacial ace-
tic, and finally 2 mL butan-1-ol. It is necessary to ensure thorough mixing of the
sample by using a vortex mixer after the addition of each component of the sol-
vent (
see
Note 15
).
2.
The 3 mL of sample is applied to the CF-1 column and its containment vessel
washed with 2 x 1 mL of fresh 4:1:1 eluant, which is also loaded onto the column
after the initial sample load has run to waste.
3.
6
3 mL of 4:1:1 eluant are now run through the column resulting in the elution
of the bulk of the standard amino acids while the collagen crosslinks remain
adsorbed to the cellulose. This portion of the eluate can therefore be discarded.
×
4.
After the 4:1:1 eluant has passed through the column, a collection vessel is placed
under the Pasteur pipette and 3
3 mL of water passed through the column to
desorb the crosslinks from the cellulose. This aqueous eluate should now be taken
to dryness (
see
Note 16
).
×
3.6. Ion-Exchange Chromatography with Ninhydrin Detection
1.
The freeze-dried aqueous phase eluate from the CF-1 column is reconstituted in
120
L of 0.01
M
hydrochloric acid by thorough vortex mixing of the tube to
ensure complete dispersion of the solution around the walls of the vessel.
µ
2.
The tube is then centrifuged briefly (30 s) to bring the solution to the bottom of
the tube and thus ensure maximum recovery.
3.
The sample should be passed through a 4 or 13 mm 0.2-
m PVDF syringe filter
to remove particulate matter. The sample is now ready for analysis on the amino
acid analyzer (
see
Note 17
).
µ
4.
The analytical column used for the analysis is the high resolution column sup-
plied by Pharmacia (Uppsala, Sweden) measuring 270
4 mm and filled with
their UltroPac 8 resin in the sodium form. The column should be maintained at
90
×
°
C throughout the analysis.
5.
Prior to application of the sample, the column should be equilibrated in 0.2
M
sodium citrate buffer pH 4.25.
6.
After the sample has been applied, the column should be eluted with 0.4
M
sodium
citrate buffer pH 5.25 (
see
Note 18
) for 46 min during which time data are col-
lected (
see
Note 19
). The column is then washed for 6 min in 0.4
M
sodium
hydroxide and regenerated for 23 min in 0.2
M
sodium citrate buffer pH 4.25
when it is ready for running the next sample.
7.
At the completion of the run, the area of each peak is computed from the col-
lected data and the concentration of each crosslink is determined by comparison
with the peak area of a leucine external standard of known concentration (
see
Note 20
for a detailed explanation of the calculations).
8.
A typical elution profile using authentic collagen crosslinking amino acids is
shown in
Fig. 2
.
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