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.
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.
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.
The required emission filters are 530BP30 for Cy2, 605DF50 for Cy3, and 695DF55 for Cy5.
Loading the Gels Into the Molecula