Differential in gel electrophoresis (DIGE)
- image analysis with Delta2D
DIGE - Differential in Gel Electrophoresis - allows for simultaneous
separation of up to three samples on one gel, bringing a new level of
statistical confidence and reliability to 2D gel electrophoresis.
Differential in Gel Electrophoresis was first described in:
Difference gel electrophoresis: a single gel method for detecting changes
in protein extracts. Unlu M, Morgan ME, Minden JS.
Electrophoresis. 1997 Oct;18(11):2071-7
Fluorescent dyes are covalently bound to the proteins prior the 2D gel
electrophoresis process. Multiple samples labelled with different dyes are
then co-migrated on the same gel. The fluorescent dyes are designed in a
way that they minimally influence the migration behavior of the proteins
and allow for the detection of lowest quantities. The unchanged protein
properties make it possible that protein patterns labeled with DIGE
fluorescent dyes can be compared also to formerly generated protein
patterns stained with Coomassie, Silver nitrate or Sypro Ruby. That means
that proteome
maps generated with Delta2D can also be used for DIGE experiments.
The main advantages of DIGE are:
- Having multiple samples co-migrate on the same gel means that
running differences are eliminated for these samples.
- Using an internal standard can compensate for experimental variation in spot quantities.
Delta2D supports this and other multiplex
gel electrophoresis techniques. It leverages advanced image processing
algorithms like image fusion to let you use internal standards, produce
complete expression profiles, and visualize the results - all in the same
package that you also use for the "classical" 2DE
experiments.
Equipment and consumables necessary for DIGE
experiments
The most important device for doing DIGE analysis is a compatible
fluorescence scanner or a camera system
for detecting the DIGE fluorescent dyes. At the moment
several scanners are available on the market which are all supported by
Delta2D. We recommend using the
Fuji FLA 5100 that meets the current needs for a DIGE scanner but is
prepared for future developments - such as dyes that are excited at
infrared wavelengths currently under development.
Additional cosumables like the fluorescent
dyes for doing DIGE experiments are necessary.
The dyes covalently bind the protein molecules and enable
for a simultanous separation of up to three differently labeled samples
per gel.
Please contact DECODON or
Fuji Photo Film
(Japan and Europe) if you are interested in a comprehensive
solution for analyzing DIGE type experiments.
One way of using the co-migration of multiple samples on one gel is to
put one sample on each of the gels in an experiment. Typically, this is a
pooling of all samples involved, called the "internal standard".
Then a spot's quantity can be divided by the quantity on the standard
image, giving greater reproducibility.
One possible experimental design, involving two samples A,B,C, is
described in the following table
Design for a DIGE experiment involving two
samples
|
Gel 1
|
Gel 2
|
Gel 3
|
Gel 4
|
Dye 1
|
Sample A
|
Sample A |
Sample B |
Sample B
|
Dye 2
|
Sample B |
Sample B |
Sample A |
Sample A |
Dye 3
|
internal standard S
|
internal standard S |
internal standard S |
internal standard S |
This design produces 12 images, four each for samples A and B, and four
for the internal standard.
When setting up a gel image analysis project using Delta2D, for each image
the corresponding internal standard is recorded. Gel
images from the same gel do not need to be warp transformed, and
quantitation is done in relation to spot quantities on the internal
standard image.
Image Fusion To ensure the detection of all spots within an
experiment on all gels we use the union image
fusion algorithm that was developed for Delta2D. The result shows a
gel image containing all protein spots occurring on any of the gels. With
the help of this image spot detection is performed, resulting in complete
expression profiles for every spot in your experiment.
Delta2D's reproducible spot editing gives you fine control over the
spot detection result while saving you from the arduous process of
painting spots by hand. You can now create, split, and join spots with
minimal user input. This not only saves time, it supports reproducibility
of your experiments because each spot correction on the fusion gel affects
the spot recognition on all gel images of the whole experiment.
No spot painting means no subjective judgement - and that means good
lab practice is maintained right through the whole analysis.
Spot Transfer During the next analysis step the predicted spot
locations from the fusion gel will be applied to all gel images of your
experiment. Appropriate warp transformes will be considered when
necessary.
Quantitative Analysis will be done at the end of the analysis
workflow. This includes spot quantitation, spot normalization considering
the internal DIGE standard and performing statistics. Spots with
interesting expression behavior may be filtered by using the filter
feature of Delta2D or may be visualized by using the extremly illustrative
color
coding approach.
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