2D Fluorescence Difference Gel Electrophoresis (DIGE)

Creative Proteomics provides DIGE service which allows for simultaneous separation of up to three samples on one gel, bringing a new level of statistical confidence and reliability to 2D 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 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.

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.

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