Biology Reference
In-Depth Information
For everyday work, dyes were dissolved in anhydrous DMF at a
fi nal concentration of 2 nmol/mL. This solution was distributed
into smaller aliquots (usually 25-30 mL) and stored in 0.5-mL
Eppendorf tubes at −80°C. Before closing, the tubes were fi lled
with argon gas. These aliquots were taken out of the −80°C freezer
as necessary for creating working dye solutions for protein label-
ing. Before returning the remaining aliquots to the freezer, the
tubes were fi lled with argon gas again.
3.3.2. Dyes Dilution
and Storage
To evaluate the effects of long-term storage of dry dyes on their
performance in DIGE, as well as the effect of dilution in DMF on
dye stability, we compared two groups of dye samples. The fi rst
group of newly synthesized dyes (Cy2, Cy3, and Cy5) was placed
in storage immediately after synthesis and stored at −20°C as dry
solids for 5 years under argon atmosphere, as described in
Subheading
3.3.1
. These tubes were taken out of the freezer and
dissolved in fresh anhydrous DMF right before being used in a
DIGE experiment. This dilution is further called NEW dilution,
and it was the same dilution used to compare newly synthesized
dyes with commercially available ones, as described in
Subheading
3.2
above. The second group of new dyes was diluted
in DMF at 2 nmol/mL at the time of dye synthesis and then stored
in DMF for 5 years, as described in Subheading
3.3.2
. This sample
is further called OLD dilution and was used to compare to the
freshly made NEW dilution in DIGE experiments.
OLD and NEW dyes dilutions were used for protein labeling
in the same type of experiment, as described in Subheading
3.2
above, except that it was done on a slightly smaller scale: instead of
fi ve pairs of samples, only three WT and three Fis
−
protein samples
were used (
N
= 3). As previously, after labeling, IEF and SDS-
PAGE separation, protein spots were analyzed using DeCyder soft-
ware in an automated fashion. Spot detection and automatic
matching was performed without manual intervention, and a pro-
tein fi lter was applied as described in Subheading
3.2.4
. Out of 25
protein spots that satisfi ed the selection criteria, 16 are upregu-
lated, and 9 are downregulated in Fis
−
cells, as compared to WT
cells (see Fig.
6
).
Average ratios in protein abundance for all 25 spots plotted in
Fig.
6
shows that selected spots in both OLD and NEW dilution-
labeled samples are very similar. As presented in Table
3
, the majority
of protein spots (21 spots, 84% of total) in both OLD and NEW
dilution samples differ by less than 10% from each other.
In summary, three dyes used in DIGE, benzoxazolium dye
Cy2, propyl Cy3, and ethyl Cy5, and their NHS esters were
synthesized from commercially available precursors. Newly synthe-
sized dyes were tested in DIGE experiments and were shown to be
indistinguishable from commercially available dyes at the level of
individual protein abundance measurements. Synthesized dyes
3.3.3. Long-Term Storage
and Dye Stability
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