Proteomics/Protein Separations- Electrophoresis/Differential in Gel Electrophoresis(DIGE)

Theory
Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This is done in three steps. First, samples are tagged with unique fluorescent dyes. Second, they are run together on the same 2D-PAGE gel. Finally, after the run completes, the different fluorescent images of the same gel are superimposed over each other. DIGE allows the study of proteins that are expressed differentially, as well as those that are common between samples. This technology allows for simultaneous separation and comparison of up to three samples on one gel.

DIGE vs. 2D-PAGE
In traditional 2D-PAGE, different samples are often separated in multiple gels. Those gels are then overlapped to compare one gel to another. Due to differences between gels in spatial resolution and spot intensities, the overlaying of images and correct matching of proteins is difficult. There can also be variations in protein uptake by the isoelectric focusing strips, incomplete protein transfer from the first to the second dimension gel, and local inconsistencies in gel composition, field strength or pH gradients. These gel-to-gel variations can mask the biological variation between the samples. Unfortunately, not all of these variations are avoidable; they can occur for a number of reasons. Thus, quantitative comparisons of protein expression levels are difficult using 2D-PAGE.

In DIGE, protein mixtures are pre-labeled, prior to electrophoresis, with cyanine dyes that guarantee co-migration of proteins. This co-migration on the same gel eliminates running differences between samples. In addition, the samples are subjected to the same environment and the same procedures throughout the experiment. Experimental variation is minimized in this way. In DIGE, there is no need to process a gel for visualization after electrophoresis.

Dyes
The cyanine dyes (Cy2, Cy3 and Cy5) used for DIGE are N-hydroxy succinimidyl ester derivatives, are covalently tagged to the ε-amino group of lysine residue of proteins, and replace the ε-amino group positive charge with the positive charge of the dye. The binding of the dyes introduce small but matched increases in molecular weight to the protein. Since these dyes are hydrophobic, to prevent precipitation of proteins, only one lysine residue per protein is labeled (minimal labeling). Minimal labeling limits the fluorescence intensity and thus the sensitivity of the stain. The dyes are all charge-matched and molecular mass-matched to prevent alterations of the isoelectric point, and to minimize dye-induced shifting of labeled proteins during electrophoresis.

There is also another class of cyanine dyes, called saturation dyes, which saturate cysteine residues instead of minimally labeling lysine residues. Again, these dyes are mass and charge-matched; but there is a smaller chance of protein modification and isoelectric point shift. Saturation cysteine dyes have superior sensitivity to minimal lysine labeling. As with the minimal lysine labelling cyanine dyes, if a cysteine amino acid is not present in a protein, then the saturation dyes will not be able to label it, leaving the protein undetectable.

Two dyes
Two protein samples are first labeled with propyl-Cy3 and methyl-Cy5. Once the samples are labeled, they are applied onto a single 2D-GE gel. After the run, the corresponding protein spot are visualized by successively illuminating the gel with the excitation wavelengths of each of the dyes. The resulting protein spots are analyzed using imaging software. Differentially expressed protein spots are then excised from the gel and identified by mass spectrometry.

Three dyes
To study multiple samples, a large number of pairwise comparisons or analysis of samples run on different gels is needed. An important alternative is to use a third dye (Cy2) with similar characteristics as Cy3 and Cy5. Protein samples are labeled with either Cy3 or Cy5, and a pooled sample containing equal amounts of all samples in the experiment is labeled with Cy2. All samples are then applied on the same gel. The pooled sample acts as an internal standard for every protein spot on each of the gels, and is then used for normalization of all spots, across all gels. This approach reduces the experimental variation, increasing both the accuracy of quantification and the statistical confidence of protein expression differences.

Advantages
To study differential expression of proteins, there is no need to run multiple gels. Loss of proteins even in the low molecular weight range is reduced since no post-electrophoretic processing (fixation or destaining) is necessary. This method is more quantitative than the standard colorimetric staining methods, both with regard to sensitivity as well as linearity.