Biology Reference
In-Depth Information
achieved by running 12.5% gels for approximately 16 h at
2.2 W per gel.
11. Images are best acquired directly after the 2DE by scanning
gels between glass plates using a Typhoon 9400 Imager or a
similar device. Ensure that both outer plate surfaces are clean
and dry before scanning and that the bonded plate is the lower
plate on the scanner bed.
12. Perform an initial low-resolution scan (1,000 mm) for one gel
on the Cy3 and Cy5 channels with the photomultiplier tube
(PMT) voltages set low (e.g., 500 V). The excitation/emission
wavelengths for fl uorescence detection using the Typhoon
9400 are 532/580 nm for ICy3 and 633/680 nm for ICy5.
An image is then built up by the scanner for each channel and
is converted to grayscale pixel values.
13. Using ImageQuant software for the Typhoon 9400, establish
maximum pixel values in various user-defi ned, spot-rich regions
of each image, and adjust the PMT voltages for a second
low-resolution scan to give similar maximum pixel values
(within 10%) on each channel and without saturating the sig-
nal from the most intense peaks. Repeat scans may be required
until values are within 10% for the two channels (see Note 9).
14. Once set for the fi rst gel, use the same PMT voltages for the
whole set of gels scanning at 100-mm resolution. A 24- × 20-cm
gel image takes approximately 10 min to acquire per channel
and two gels are scanned simultaneously. Images are generated
as gel fi les.
15. Crop overlayed images in ImageQuant and import into
DeCyder Batch Analysis software for subsequent BVA analysis,
according to the DeCyder software user manual. An example
of using ICy to monitor H
2
O
2
-induced redox-proteome alter-
ations in HMLECs is shown in Fig.
4
. A number of proteins
showed a rapid increase in labeling (e.g., spots 25 and 26),
suggesting generation of new free thiols, for example, via scission
of disulfi des. On the other hand, a number of proteins showed
a rapid decrease in labeling (e.g., spots 24 and 27), implying
oxidation of free thiols that would not be labeled with the ICy
dyes. In some cases, the change in ICy labeling recovered over
time, indicating reversible modifi cation (e.g., spot 27) (see
Note 10).
1.
After ICy fl uorescence image scanning, gels are immersed in
fi xing solution and incubated overnight with gentle shaking.
Fixed and bonded gels can now be stored for several months at
4°C by sealing in plastic bags with 1% (v/v) acetic acid.
3.5. Post-Staining
and Spot Excision
2.
For post-staining, the colloidal Coomassie Brilliant Blue (CCB)
G-250 staining method is modifi ed from that of (
20
). Fix gels
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