Multifluorescence 2D Gel Imaging and Image Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 24

performed in one pass without interruptions and without changing
scan parameters. In case of using a laser fl uorescence scanner for
gel imaging, the imaging procedure for a set of twelve large 2D
gels may take several hours, so electrophoresis should run over
night and has to be terminated early in the morning. After removing
the gel cassettes from the electrophoresis chamber, a fi rst washing
step is carried out with deionized tap water to remove remains of
running buffer. To prevent the gels from drying and shrinkage,
they should be left in their cassettes until scanning. The washed
cassettes can be stored as stacks in shaded plastic boxes together
with wetted lint-free paper. Because fl uorescence intensity is tem-
perature dependent, all gels should be scanned at a defi nite tempera-
ture ( 2 ) (see Note 1).
In case of using a laser scanner with confocal optics, the gels
will remain in gel cassettes even during the scanning procedure. To
prevent the formation of Newton's rings, gel cassettes should not
be placed directly on the scanners' glass platen, but on a holding
tray that determines a small gap between the bottom of the cas-
sette and the top of the platen. Immediately before scanning, gel
cassettes have to be cleaned meticulously for a second time with
highly pure water and should be dried subsequently with lint-free
paper. The cleanliness of the scanner platen surface should be
checked prior to each scanning procedure. Cleaning should be car-
ried out using lint-free tissue soaked in highly pure water. In the
long run, remains of fl uorescence dyes may cause streaky scanning
artefacts which may render the image useless for later analysis. To
prevent such streaks, the gel cassettes and glass platen have to be
cleaned at regular intervals with 10% hydrogen peroxide followed
by a washing step with highly pure water (see Note 2).

To ensure the operational readiness of the imaging system, it
should be turned on about half an hour before the fi rst gel scan is
performed. As mentioned above, all gels of one MFA experiment
should be scanned in one single “scanning session” without any
intermediate change of the scan parameters (see Note 3).
After the completion of each scan, the technical quality of the
generated image should be verifi ed before the gel is removed from
the scanning device. In case of poor image quality or scan errors, it
would be possible to readjust the scanner and to image the gel for
a second time. Otherwise, the gel can be transferred to the fi xing
solution.
Besides the practical aspects of gel handling, special attention
should be paid to the appropriate setting of the scan parameters.
Some crucial parameters for multifl uorescence 2D gel imaging will
be discussed in detail below.

3.2.3. Gel Scanning
and Scan Parameters

Depending on gel size and whether the gels are scanned in gel
cassettes or not, some gel-specifi c settings have to be adjusted.

Gel-Specifi c Settings