Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 386

The pellet may be either directly dissolved in DIGE
labeling buffer or lyophilized fi rst (see Notes 51 and 52).
Try to work out the best cell count-to-buffer volume ratio
for your type of specimen.
Tissues/organs: Depending on its rigidity, the minced tissues -

may either be dissolved in DIGE labeling buffer, or homog-
enized in it (e.g., using a Potter-Elvehjem homogenizer
or manually, using a sample grinding kit; GE Healthcare;
see Notes 53 and 54). Ultrasonication as well as freezing/
thawing support solubilization.
For all samples with variable protein content, it is necessary -

to determine the protein concentration. The easiest way to
do this is a Coomassie G-based photometric protein deter-
mination according to Bradford ( 9 ), e.g., optimized
for microplate readers. Samples may be diluted with water
to avoid quenching through additives such as urea and
detergents in the assay (see Note 55).

2. Sample application: Sample application as described here, i.e.,

cup loading, allows applying the sample at a chosen position—
away from the bulk of proteins in your sample or at a pH most
benefi cial (or least degrading) for your proteins. However, for
DIGE application, this means that a sample with a high protein
and a considerably low salt concentration is needed. Body
fluids other than serum may be lyophilized and dissolved in
a small volume of DIGE labeling buffer. Cellular proteins,
especially with hydrophobic properties, are often soluble only
to a small extent and in low concentrations and, thus, in-gel
rehydration (active or passive, i.e., with or without current)
seems advisable (see Note 56).

3. Alkaline proteins need special care (and equipment): running

under oil to protect from oxidation, additional reducing agent,
and usually shorter runs and anodic sample application (for
details see ( 10 )). When using alkaline pH gradients in the IPG

strips, lower dye-to-protein ratios may be chosen to avoid
staining artifacts (over-labeling).
4. There are several modifi cations of 2DE, e.g., without reduction
or using different additives (for a review see ( 10 )). In addition,
even when using a standard 2DE system, pH range and gel pore
size (in SDS-PAGE) may be adapted to be able to either get a
good general overview or to zoom into different parts of the
proteome ( 11-13 ). For homemade and hand-cut IPG strips, the
strip width may be varied in order to increase sample volume.
5. A larger gel system (Ettan-DALT) is described in detail in ( 8 )

including commercial IPG strips of 24 cm-length (pH 4-7 and
6-9) with 7 M urea and 2 M thiourea and large size homogenous
SDS-PAGE in the second dimension. As for sample preparation,
TCA precipitation is described for dilute protein samples.