CyDye DIGE Fluor minimal dyes are the basis of the two dimensional-differential in gel electrophoresis (2D-DIGE) technology. All three CyDye DIGE Fluor minimal dyes (Cy2, Cy3 and Cy5) have the same mass and charge and can be segregated spectrally. The distinct signal from each CyDye label plays a part in the high accuracy of 2D-DIGE analysis. An amide linkage links the NHS-ester reactive group of the CyDye to the lysine residue of the protein. The amount of labeling is controlled by the ratio of CyDye to protein in the labeling reaction. Thus, it is possible to label a single lysine residue per protein (i.e. minimal labeling). Also, the pI of the labeled protein is not significantly altered as compared to the same unlabelled protein.
There is minimal loss of signal during labeling, 2D separation and the often laborious scanning. The specific chemistry of the three dyes allows for multiplexing of three complex protein mixtures on to one 2D gel. The two protein samples to be compared along with an internal standard (usually a pool of all the samples) are individually labeled with one of the CyDyes. Post-labeling, the samples are combined and separated by isoelectric focusing followed by SDS-PAGE. The ability to separate the proteins from three different samples at the same time by isoelectric focusing and SDS-PAGE is core to the DIGE technology. This makes it possible for the same protein from the three samples to migrate to the same position on a 2D gel.
This unique experimental design is benefited by the use of DeCyder™ Software. DeCyder™ spot detection is based on initial detection of the protein spots in the Cy2 image of pooled sample (internal standard) followed by the application of similar spot boundaries to the remaining images (Cy3 and Cy5) within each gel. The internal standard improves matching intra- and inter-gel images, thereby allowing accurate quantification and separation of biological variation from experimental variation. The generation of identical spot detection patterns on all of the images from a gel is made possible by the powerful algorithm of DeCyder™ Software.
The internal standard as well as the multiplexing capability of the CyDye DIGE Fluor minimal dyes virtually eliminates gel-to-gel variation, thereby making the qualitative and quantitative image analysis more robust. The linear dynamic range for CyDye is five orders of magnitude as compared with other traditional staining techniques which range form 2 to 4 orders of magnitude.
For optimal mass spectrometry results, I recommend staining with a total protein stain after running the 2D gel. This is due to the fact that the spots visualized on a 2D gel with CyDye labeled samples are not always the point of highest protein concentration on the gel. Most protein spots contain labeled as well as unlabeled protein. The problem of not being able to pick from the area of highest protein quantity, as it does not necessarily correspond to the visualized protein spot on a 2D gel, can be circumvented by staining the gel with total protein fluorescent stain before spot picking. With regard to labeled proteins, proteins labeled with the CyDye DIGE dyes generally contain only one label per protein; therefore, the sequence generated by mass spectrometry from a CyDye labeled protein is comparable to an unlabeled protein.
Overall, the CyDye technology has enabled comparison of a control sample with treated or diseased samples at different time points and with high confidence for expression proteomics.
Dr. Nilesh S. Tannu
Research Associate
Physiology and Pharmacology
Wake Forest University School of Medicine