2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) is a variant of two-dimensional gel electrophoresis that offers the possibility to include an internal standard so that all samples—even those run on different gels—can easily be compared and accurately quantitated.
To perform 2-D DIGE, label your protein samples and internal standard with different CyDye DIGE Fluors. Run the internal standard, and up to two samples, on each gel. Using an internal standard—and running multiple samples on the same gel—ultimately means fewer gels, less manual labor, and significant cost savings.
The picture to the right is a 2-D DIGE protein spot map.
- An internal standard that virtually eliminates experimental gel-to-gel variation. No technical replicates need to be run to confirm differences in protein abundance.
- Increased throughput and significantly reduced analysis time and cost.
- Dependable results with far fewer 2-D gels (multiplexing two samples per gel with internal standards).
- Detection of true differences in protein expression with extremely high statistical confidence.
The internal standard is made by pooling all samples in an experiment, and is run on every 2-D DIGE gel. This means that there is a standard for every spot on the gel, and that all gels within the same experiment are quantitatively linked. When you have several samples, 2-D electrophoresis can be cumbersome due to the need for large numbers of replicates. Because the internal standard virtually eliminates gel-to-gel variation, technical replicates are not necessary when you perform 2-D DIGE.
The first step is labeling your protein sample with fluorophores. While proteins are post-stained in traditional 2-D electrophoresis, the protein labeling step in 2-D DIGE is performed before electrophoresis using CyDye DIGE Fluors. First and second dimension electrophoresis is performed similarly to traditional 2-D electrophoresis, for example using an IEF system, IPG strips, and an electrophoresis system.
Image analysis of 2-D DIGE gels is performed with a biomolecular imager that detects multiplex fluorescence such as Typhoon FLA 9000. Analysis is performed using DeCyder 2-D Differential Analysis Software, optimized for 2-D DIGE.