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Implementation of
the DIGE technique for Relative Expression Levels of Gel-Separated Proteins
As a result
of the expressed need on the part of SCCPIR members, the Core has recruited
an assistant director who is an expert in the areas of protein quantitation
using differential gel expression analysis (DIGE), and the Core will be
greatly expanding its capabilities in this area. DIGE is a technology
developed and marketed by Amersham Biosciences. It is centered on the
use of three cyanine dyes or labels that are matched in molecular mass
and charge. The labels are fluorescent and have non-overlapping excitation
and emission maxima. In a typical DIGE experiment the samples and control
are each labeled with only one of the three dyes. After labeling, the
samples and control are pooled, loaded onto a single IPG strip and separated
first by isoelectric focusing then SDS-PAGE. A fluorescent imager is then
used to create an image of the proteins contained in each preparation
(i.e., sample or control. Selection of appropriate filters for a particular
cyanine label allows each protein preparation to be imaged independently.
Software is then used to overlay the images, define spot boundaries and
compare quantitation levels. The main benefit of DIGE technology is the
ease in comparison of the protein preparations since the samples and control
are run on the same gel and are separated under the same electrophoretic
conditions. This is a major shift from what the Core has offered in the
past, where the collaborator runs the gel in his own laboratory, and brings
it to the facility for mass spectrometric identification of the separated
proteins. With this new technology in the Core Facility, SCCPIR researchers
will be able to more clearly and easily detect changes in protein expression
levels, and will then be able to select these proteins for mass spectrometry-based
identification.
Presentation
by Christoph Borchers to SCCPIR members (13MB)
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