DU

Differential In Gel
Electrophoresis
Differential In Gel
expression
Electrophoresis (DIGE) allows an investigator to
level of proteins reliable compare the
labeling ofa sample with thebetween
two samples on a 2D
gel platform. Limited lysine
two samples y3, cy5 and cy2 dyes allows the reliable
utilizing an internal standard for normalizing comparison of
the relative abundance
protein. Samples are labeled separately and then
to mixed to allow resolution on a of each
minimize experimental variation and improve spot single gel
proteins span thè linear range of 0. 125ng to matching. Detection level of
lower than amounts 10ug, allowing detection of protein levels
necessary for MS identification.
Saturation
be used when
examining low abundance proteins. Comparison oflabeling oniscysteines can
the DeCyder software which
allows studies from a single as well as samples done with
statistical data from a series of gel generating
gels to increase the reliance. The Proteomic
has all the and
reagents equipment necessary to perform DIGE Analysis Core
information regarding the use of DIGE can experiments.Additional
be viewed at the Amersham web site
(http://www4.amershambiosciences.com).
and an example of
Below is a flow sheet of the DIGE
protocol
DeCyder analysis done on samples in the PAC.
The sample shown below were labeled with cy5 and untreated
were mixed and resolved in the first dimension on a samples with cy3. Samples
SDS 8-16% PAGE. pH gradient of 4-7, followed by a
Samples were visualized with the Typhoon laser scanner followed by
analyzed with the DeCyder software package. Upper left is enlarge area of
single spot that with a>2 volume increase. Upper right is gel showing a
graphic representation
red depicts all spots with a >2
decrease, blue >2 increase. 88% of protein showedofnotdata,
shown on graph were showed no
Lower right is a table with all
change. Lower left is a volume representation of spot.
pertinent data of each spot.

is
Sample labeled with ey3
Sample labeled with cy5

Samples combined and resolved on 2D gel

Scanned for cy3 and cy5 on separate channels

Differential expression determined with

DeCyder software package
tinib inel qel

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FDed D

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Spots of interest processed for MS analysis