Difference Gel Electrophoresis
Difference Gel Electrophoresis (DIGE) allows the separation of treated (or diseased) and untreated (or control) samples in a single physical gel. It gives an opportunity for quick comparison in the differences of the protein profiles of each sample by overlaying the unwarped maps of treated and untreated samples. It is possible to see which proteins are shared by both, which are present in one sample but not in the other. In a DIGE system, proteins are pre-labelled with fluorescent CyDyes™ such as Cy2, Cy3, and Cy5 prior to electrophoretic separations. Labelled samples are then mixed before isoelectric focusing, and resolved on the same 2D gel. Cy2 dye is used to label an internal standard, which consists of a pooled sample comprising of equal amounts of each of the samples to be compared. This allows both inter and intra gel matching, and is used in the standardization of spot volumes in different gels. Spot volumes are expressed as a ratio to the internal standard. Images of each dye are acquired with various lasers using a variable mode imager and images are analysed with differential image analysis software. DIGE provides far more confidence with the level of accuracy (no false negative and no false positive) and saves time by reducing the large number of replicates that are used in the conventional, single stain 2D gel method. If absolute biological variation between samples is the primary target, then DIGE is the method of choice.
Sensitivity of CyDyes
Cy2 - 0.075 ng
Cy3 - 0.025 ng
Cy5 - 0.025 ng
Key benefits of DIGE:
- Multiplexing, internal standard, less gels, quantitative data and high accuracy.
- Confidence in the results reflects true biological outcomes and is not due to the technical variation.
Human plasma DIGE gel, IPG: 4-7, second dimension 8-18%
Saturation CyDyes label all the available cysteine residues. This technique is suitable for scarce samples as excellent arrays can be produced with only 5μg of protein per channel. There are two size and charge matched Saturation CyDyes available, Cy3 and Cy5.
Human plasma Saturation DIGE gel, IPG: 4-7, second dimension 8-18%, 5μg per sample
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