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Difference gel electrophoresis (DIGE) is a relatively straightforward
application of differential labeling of protein samples using fluorescent
dyes. The technique, originally published by Unlu et al. (1997), uses
cyanine (Cy) dyes to differentially label proteins separated by 2-D gel
electrophoresis. Following labeling and electrophoresis, excellent results
can be achieved by scanning the multiplex gels with the Molecular Imager
FX multiimager system and analyzing them with PDQuest 2-D analysis software.
The DIGE method is designed to compare two complex protein samples derived from two different growth conditions. One sample is labeled with Cy3 and the other with Cy5. A third dye, Cy2, is used to label a 50:50 mixture of the two samples, which becomes a standard for registering the images obtained with the other two dyes. Equal amounts of the three labeled protein mixtures are combined and run on the same 2-D polyacrylamide gel, eliminating any gel-to-gel variation.
Imaging
Image Acquisition With the Molecular Imager FX System
The Molecular Imager FX multiimager systems are well suited for image
acquisition from gels containing Cy-dyed proteins. The laser and filter
combinations provided ensure minimal cross-talk between the different
fluors.
Lasers
The lasers required for the Cy dyes are the 488 nm external laser for
Cy2, the 532 nm internal laser for Cy3, and the 635 nm external laser
for Cy5.
Emission Filters
The required emission filters are 530BP30 for Cy2, 605DF50 for Cy3, and
695DF55 for Cy5.
Loading the Gels Into the Molecula
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