The Basics of 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 10

ampholytes to establish the pH gradient). Despite this increase in
reproducibility, there were still problems with gel-to-gel reproduc-
ibility requiring the use of technical replicates to remove artifacts
of experimental variation. This can result in a prohibitive number
of gels, especially for complex experimental designs.
A breakthrough in 2D gel electrophoresis arrived with the
introduction of the ability to multiplex fl uorescently labeled pro-
teins on the same gel ( 4 ). This technique is referred to as 2D dif-
ference gel electrophoresis (2D DIGE) (see reviews (refs. 5-7 )).
The fl uorescent dyes used are specially modifi ed cyanine dyes
(CyDye™ DIGE fl uors) which are matched for molecular weight
and charge and provide a useable dynamic range of up to 4 orders
of magnitude. There are two approaches to the labeling; the most
common approach is termed minimal labeling, where the dye binds
to a restricted number of lysine residues. For certain samples an
alternative approach termed saturation labeling is used, where the
dyes bind to all of the accessible cysteine residues.
The minimal dyes (Cy™2, Cy3, and Cy5) all have an approxi-
mate molecular weight of 450 Da and carry a +1 charge (this
replaces the +1 charge of the lysine resulting in no overall change
to the pI). The dye-to-protein ratio is controlled such that only a
small percentage of the total available lysine population is labeled
with the CyDye to avoid multiple labels per protein. By utilizing
size- and charge-matched dyes, the labeled proteins will comigrate
on the 2D gel and allow precise image overlay from each sample.
For the saturation dyes (Cy3 and Cy5) the opposite strategy is
used for labeling. The dye and reductant concentrations are opti-
mized to ensure that all the reduced cysteine residues are labeled
with the CyDye, resulting in an increase in signal. These dyes have
a molecular weight of 680 Da and are neutrally charged. Samples
labeled with saturation dyes will exhibit altered spot migrations
due to the number of cysteines present and will thus display a dif-
ferent spot pattern compared to the minimal labeling approach.
However, the samples within the same gel will comigrate such that
differential analysis can still be performed.
Running differently labeled samples in a single gel and analyz-
ing the resulting images can provide possible proteins of interest.
However, to allow for biological variation the use of biological
replicates for statistical confi dence is necessary, so multiple gels still
need to be run. To overcome the problems of gel-to-gel variation,
one of the dyes is used to label a pooled internal standard (some-
times referred to as a pooled internal reference) (see Fig. 1 ). The
pooled internal standard is comprised of all the potential detect-
able proteins in the experiment such that it is a combination of
equal aliquots of each of the samples to be analyzed (see Table 1 ).
The virtual elimination of gel-to-gel variation (coupled with
multiplexing) now allows for the running of biological replicates
such that the number of gels to be run is dramatically reduced