Proteomics/Protein Separations- Electrophoresis/Types of Gel Electrophoresis/Differential in Gel Electrophoresis
Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This is done by fluorescent tagging of two samples with two different dyes, then they are run the same 2D- PAGE, after the run superimpose the two flourescent images of the same gel to study proteins common in both samples and also proteins that are expressed differentially. This technology allows for simultaneous separation and comparison of up to three samples on one gel.
DIGE vs 2D- PAGE
In the traditional 2D-PAGE to compare differential expression of proteins, different samples are often separated in multiple gels and then overlapped in order to compare different spots or intensity of spot changes from one gel to another. Due to differences between gels in spatial resolution and spot intensities the overlaying of images and correct matching of proteins difficult. These are also gel-to-gel variations in protein uptake by the isoelectric focusing strips, incomplete protein transfer from the first to the second dimension gel and local in homogeneities in gel composition, field strength or pH gradients. These gel-to-gel variation are the experimental variations and that masks the biological variation between the samples. Thus quantitative comparisons of protein expression levels are difficult using 2D - PAGE.
In DIGE protein mixtures are pre-labelled with cyanine dyes that guarantee co- migration of proteins, prior to electrophoresis. This co-migration on the same gel eliminate running differences these samples. In addition, the samples are subjected to same enviornment and procedures throughout the experiment,so it minimizes the experimental variation in spot quantities. In DIGE, there is no need for post-processing of gel for visualization.
The cyanine dyes (Cy2, Cy3 and Cy5) used for DIGE are N-hydroxy succinimidyl ester derivatives,are covalently tagged to the amino group of lysine residue of proteins and replaces the -amino group positive charge with the positive charge of the dye. The binding of the dyes introduce small but matched increase in molecular weight. Since these dyes are hydrophobic, to prevent precipitation of proteins only one lysine residue per protein is labelled (minimal labelling). 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 pI, and minimise dye-induced shifting of labelled proteins during electrophoresis.
Another approach is to use cyanine dyes other than the previously described dyes, which saturate cysteine residues instead of minimally labelling lysine residues. Again these cyanine dyes used are mass and charged matched; but there is a smaller chance of protein modification and pI shift. Saturation cysteine dyes have superior sensitivity than 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 undectatable.
- Using 2 dyes
Two protein samples are first labeled with propyl-Cy3 and methyl-Cy5. The labelled samples are then mixed and applied onto a single 2D-GE gel and after the run the corresponding protein spot patterns are visualized by successively illuminating the gel with the excitation wavelengths of each of the dyes.To view images click here[][] The resulting protein spots are analyzed using software. Differentially expressed protein spots are then excised from the gel and identified by mass spectrometry.
- Using 3dyes
Only a direct comparison of two different protein samples can be performed using two dyes. To study multiple samples, a large number of pair wise 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 labelled with either Cy3 or Cy5 and a pooled sample containing equal amounts of all samples in the experiment is labelled 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 and increases the accuracy of quantification and the statistical confidence of protein expression differences.
To study differential expression of proteins, no need to run mutliple 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.Also differences in spot fluorescence intensity are purely attributable to biological and not experimental variation with increased confidence.
- Mustafa Ünlü , Mary E. Morgan , Dr. Jonathan S. Minden:Difference Gel Electrophoresis: A single method for detecting changes in protein exteacts;Electrophoresis(1997) 18:2071-2077
- Gert Van den Bergh,Lutgarde Arckens:Fluorescent two-dimensional difference gel electrphoresis unveils the potential of gel-based proteomics ;Current Opinion in Biotechnology(2003) 15:38-43