Biology Reference
In-Depth Information
out covering the slot, to make the connection between the
electrode and the gel. The anode electrode wick is similarly
placed on the other side of the gel.
2. As for the vertical system, the IPG strips are incubated in equil-
ibration buffer containing 0.3 % (w/v) urea and 0.8 % DTT
(w/v) for 15 min and then a second step with the equilibra-
tion buffer complemented with 0.3 % (w/v) urea and 2 %
(w/v) IAA.
3. After equilibration, IPG strips are deposited, gel side down, in
the strip-slot. The lid is closed on each gel-drawer, allowing
the application of the electric current.
4. The second-dimension run is started according to the manu-
facturer's instructions at 15 °C either for several hours or over-
night. The strip is removed from the gel after 70 min of
low-voltage steps (4 W for 30 min—12 W for 30 min—20 W
for 10 min for 4 gels). The immobilized pH gradient would
interfere with the high voltage of the next steps resulting in a
poor resolution. The run itself is set at 120 W for 4 h, then
160 W for 50 min for short run and 8 W overnight (10-15 h),
and then 120 W for 3 h for overnight runs. The run is stopped
when the dye front reaches the end of the gel.
1. When the gels are taken out of the SDS-PAGE tank, they are
thoroughly rinsed with water (
see
Note 10
) and dried with paper
before the image acquisition in a scanner with three different
lasers—or lasers able to combine three adequate wavelengths of
emission and excitation—(here a Typhoon imager). Images are
acquired by using three different lasers at 488, 532, and 633 nm
(excitation wavelengths) and 520, 590, and 680 nm (emission
wavelengths) for Cy2-, Cy3-, and Cy5-labelled proteins, respec-
tively (thus, three images per gel, corresponding to the two
samples and the internal standard) (
see
Note 11
).
3.5 Image
Acquisition
and Analysis
2. After image acquisition, the fi rst step for the image analysis is
the detection of the spots. Software dedicated to DIGE
image analysis is able to detect the spots simultaneously on
the three images obtained from the same gel. One of the
main advantages of the DIGE is the use of the internal stan-
dard for the gel-to-gel matching (as the same sample is run
on all the gels from the experiment) but also for the normal-
ization among the different gels. This feature helps the future
analysis of the different gels and the comparison of the pro-
tein abundances. After the gel-to-gel matching, powerful sta-
tistical analyses can be achieved on thousands of spots present
in all the gels [
15
,
16
].
3. Once proteins of interest for the experiment have been selected
on the basis of statistical analysis, the next step is the excision of
