Difference Gel Electrophoresis (DIGE) is a technology, that enables separation and quantification of fluorescent-labeled proteins (Cy2, Cy3, and Cy5) from different samples through a two dimensional gel electrophoresis. Protein samples are differentially labeled using three cyanine fluorescent dyes (Cy2, Cy3 and Cy5; Amersham Biosciences) prior to gel electrophoresis. Groups of labeled proteins are then mixed and separated by one 2D-gel. At the first dimension of the 2D-gel, proteins are separated by IEF, which is based on the isoelectric points of proteins. In the second dimension, proteins are separated in PAGE gel based on the molecular weight of proteins. Afterwards, fluorescent images at each channel that are used for detection of corresponding fluorescent-labeled proteins are captured. image from each channel is superimposed with one another, which are automatically analyzed by the DIGE software. Software can identify the significant distinct spot to be sent for mass spectrometry analysis for further quantification of the proteins in the picked spot.
Images of different channel represent different protein samples. Superimpose of these images reveal the difference between different protein samples. For example, if protein A is upregulated, the intensity of the signal of the spot, which corresponds to protein A, will be much higher. When a internal standard protein is also run in the same gel, the absolute amount of proteins contained in each sample can be calculated based on the signal intensity. In addition to protein quantification study, DIGE can also be applied to study protein post-translational modifications. The types of modification can be reflected by the shift of proteins on the gel image.
Comparison between traditional 2D-PAGE and DIGE:
|Labeling method||Fluorescent dye labeling||Gel staining|
|Number of gel||One gel||Multiple gels|
|Number of samples per gel||2 to 3||1|
|Gel to gel variations||Yes||No|
Workflow of DIGE Analysis:
1. Protein samples quantified and normalized.
2. Protein samples (optional choice for internal standard) are tagged with fluorescent dyes.
3. Protein samples are mixed and separated in one 2D-PAGE gel.
4. Fluorescent images under each channel are captured and superimposed over each other.
5. Computational analysis of the images.
Features of DIGE:
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