Figure 2: Coupling of protein with minimal cyanine dye

These dyes have unique fluorescence properties. Cy2 when excited at 492 nm gives fluorescence maxima at 510nm (green fluorescence). Cy3 when excited at 550nm give fluorescence maxima at 570nm (yellow fluorescence) and Cy5 when excited at 650nm give fluorescence maxima at 670nm (red fluorescence). Thus cyanine dyes labeling gives unique fluorescence property to proteins. When coupled to proteins, these dyes add approximately Mr 500 to the protein's mass. However, as size and mass of all dyes are matched, a protein labeled by different dye will migrate to the same position. Using these three dye at a time three samples are analyzed.

 

When dye coupling is done, concentration of dye is kept limiting which leads to 1-2% of lysine residue labeling. In this process only one dye molecule per molecule is labeled. Even if multiples lysine residues of a protein are labeled, percentage of this double labeled species is too small to be visualized. As we have discussed that the binding of these dyes do not effect isoelectric point of protein but adds approximately Mr 500 mass. Unlabeled fraction of protein corresponding to a given spot resides at Mr 500 difference where fluorescence signal of the spot is seen. Once the spot of a protein of interest is identified by image analysis, recovery of the unlabeled protein by automated spot is achieved for various analysis.