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Two dimensional difference gel electrophoresis
(DIGE) utilizes mass- and charge-matched, spectrally resolvable fluorescent
dyes (Cy3 and Cy5) to label two different protein samples in vitro prior to
2-D electrophoresis. Compared to conventional 2D gel electrophoresis, DIGE
has the major advantage that both the control and experimental sample are
run in the same gel. These samples are then imaged separately but because
they were run in the same gel, the images can be perfectly overlaid without
"warping". This reduces the number of gels that must be run to make
statistically valid comparisons and raises the confidence with which protein
changes between samples can be detected and quantified. Use of a third dye
(Cy2) permits an internal standard to be created by pooling an equal aliquot
of all biological samples in the experiment. The internal standard is then
run on every gel in the experiment. This means that every protein spot from
all samples will be represented in the internal standard. This in turn
allows more accurate quantification and spot statistics between gels. Based
on the literature, it may be possible to profile up to 1,000 protein spots
on properly prepared samples that provide well resolved 2D gels. An
additional advantage of this system is the ability to detect many
post-translational modifications, such as phosphorylation, which often play
a key role in modulating protein function and which cannot generally be
detected by ICAT-MS based protein profiling.
Additional Information on
DIGE:
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