Amersham CyDye DIGE fluorsSupplier: Cytiva
- Allow detection of up to three prelabelled protein samples and standards on the same 2-D electrophoresis gel
- Size- and charge-matched dyes enable co-migration of labelled samples within the gel
- Bright and highly sensitive dyes allow the use of the minimal labelling technique
- Minimal loss of signal during labelling, separation, and scanning
- No change in signal over wide pH range used during first-dimension (IEF) separation
- Discrete signal from each fluor with minimal cross-talk contributes to high accuracy
- Soluble in cold and hot water (solid)
Protein samples and the internal standard are each labeled with one CyDye™ DIGE fluor minimal dye. These labelled samples are then combined, run on an isoelectric focusing gel in the first dimension, and separated by SDS-PAGE in the second dimension.
The ability to multiplex different CyDye™ DIGE fluor minimal dye-labelled samples on the same gel means that the different samples will be subjected to exactly the same first- and second-dimension running conditions. Consequently, the same protein labelled with any of the CyDye™ DIGE fluor minimal dyes and separated on the same gel will migrate to the same position on the 2-D gel and overlay. This limits experimental variation and ensures accurate within-gel matching.
Protein samples and the internal standard are each labelled with one CyDye™ DIGE fluor minimal dye. These labelled samples are then combined, run on an isoelectric focusing gel in the first dimension, and separated by SDS-PAGE in the second dimension.
The NHS ester reactive group of CyDye™ DIGE fluor minimal dyes covalently attaches to the epsilon amino group of lysine of proteins via an amide linkage. The ratio of dye to protein has been designed to ensure the dyes are limiting in the reaction. As a result, approximately 3% of the available proteins are labelled and then only on a single lysine per protein (one dye per protein, or minimal labelling).
The amino acid lysine in proteins carries an intrinsic single positive charge at neutral or acidic pH. CyDye™ DIGE fluor minimal dyes also carry a single positive charge which, when coupled to the lysine, replaces the single positive charge of the lysine with its own, ensuring that the pI of the protein is not significantly altered when compared with the same unlabelled protein.