Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 382

5. Running conditions: Usually, strips are run with the following

program consisting of a series of voltage steps: 1 h at 100 V,
1 h at 200 V, 9.8 h at 500 V, 5 h at 1,000 V, and 1.5 h at
2,000 V (up to 13.2 kVh). Then the run is terminated, the
chamber opened, and the sample application pieces removed.
The strips are inspected: They should be more or less dry, with
slight “wrinkles” (corresponding to the iso-pH-lines generated
by the ampholytes). In serum, CyDye labeled albumin will be
visible as colored multiple bands. The electrodes are replaced
and the run is continued (1 h at 200 V, 1 h at 500 V, 0.5 h at
1,000 V, and 0.25 h 2,000 V, i.e., to 14.9 kVh in total). This
focusing program is slow, but has the advantage that it can
cope with higher amounts of salts or considerable protein load
(see Notes 33 and 34).

6. Immediately at the end of the run, the strips are transferred

into a transparent cover (ends fi xed with staples), labeled, and
frozen at −20°C (see Note 35). They may be stored for days/
weeks. Alternatively, they may be directly prepared for the
second dimension (see Subheading 3.5 ) if the SDS-PAGE gels
have already been prepared (see Subheading 3.4 ).

3.4. Preparation
of SDS-PAGE Gels

1. Gels are cast between two low-fl uorescence glass plates, with

1.5-mm spacers. Up to three of these assemblies fi t into the
gel-casting cassette (the name 4-gel caster given by the manu-
facturer is misleading for this gel thickness; with 1.5-mm
spacers, three gels are the limit); the rest is fi lled with glass
plates, fi ller sheets, or acrylic spacer plates (see Note 36).

2. The separation gel is cast as a gradient gel with a similar setup

as described in Subheading 3.2 but with a larger gradient
maker and a higher pumping speed. In this case, the casting
cassette is better fi lled from the bottom. First, the tubing is
fi lled with water, without trapping air bubbles, and additional
6-8 mL of water are put into the cassette, to achieve an even
and fl at gel surface. Then the gradient is started between the
light and the dense gel solution. The composition of the two
gel solutions for casting 10-15%T-gradient gels is listed in
Table 2 (see Note 37). Be careful to exclude any air bubbles
between the two reservoirs of the gradient maker and to adjust
the stirring speed during casting. After the last drops of the gel
solutions are pumped into the cassette (slightly tilt the gradient
former for this, but avoid introducing air), immediately continue
with the glycerol-bromophenol blue solution. When the appro-
priate height of the gels is reached (about 5 mm below the
upper edge of the glass plate), the pump is stopped, and the gel
left overnight at room temperature for polymerization (see
Note 38). Next morning, the cassette can be opened and the
individual gel assemblies either processed immediately or
stored at 4-6°C (see Note 39).