|
|
 |
|
 |
|
|
DIFFERENCE GEL ELECTROPHORESIS (DIGE)
Two dimensional difference gel electrophoresis (DIGE) utilizes mass- and charge-matched, spectrally resolvable fluorescent dyes (Cy3 and Cy5) to label two different protein samples in vitro prior to 2D electrophoresis. Compared to conventional 2D gel electrophoresis, DIGE has the major advantage that both the control and experimental sample are run in the same gel. These samples are then imaged separately but because they were run in the same gel, the images can be perfectly overlaid without "warping". This reduces the number of gels that must be run to make statistically valid comparisons and raises the confidence with which protein changes between samples can be detected and quantified. Use of a third dye (Cy2) permits an internal standard to be created by pooling an equal aliquot of all biological samples in the experiment. The internal standard is then run on every gel in the experiment. This means that every protein spot from all samples will be represented in the internal standard. This in turn allows more accurate quantification and spot statistics between gels. Based on the literature, it may be possible to profile up to 1,000 protein spots on properly prepared samples that provide well resolved 2D gels. An additional advantage of this system is the ability to detect many post-translational modifications, such as phosphorylation, which often play a key role in modulating protein function. Proteins are identified from another gel, termed the pick gel, which is typically overloaded and stained with Sypro or Coomassie. Spots from the DIGE gel are matched to the pick gel and a pick list is generated from the spots showing statistically significant changes.
The CNRU Mass Spectrometry Core Facility performs DIGE experiments using GE Healthcare (Amersham) reagents. Imaging is accomplished with a Typhoon Trio Imager and spots are analyzed using either DeCyder software or a software package from NonLinear Dynamics. Contact us for more information on this service.
|
|
|
|
 |
