Capillary electrophoresis with laser-induced fluorescence detection of indigo carmine and indigo carmine-labeled proteins.
Indigo carmine blue is a water-soluble, light-sensitive, anionic dye most widely applied in microscopic staining techniques. It is virtually nonfluorescent in its oxidized state in aqueous solution, but when reduced under alkaline conditions, it becomes fluorescent, with absorption and emission maxima at 436 and 528 nm, respectively. It is demonstrated that the fluorescent character of the reduced form of the dye can be exploited as a label for the determination of cationic proteins by capillary electrophoresis with laser-induced fluorescence detection. Model proteins trypsinogen and cytochrome c are noncovalently labeled with indigo carmine; the bound species are reduced, rendering the indigo carmine fluorescent; and capillary electrophoresis with laser-induced fluorescence detection is used for subsequent analysis. Various buffer systems and buffer additives were examined and optimized, and a suitable pH range for optimal fluorescence intensity and protein-dye interaction was established. Fluorescence quenching of the reduced dye when bound to protein was observed in all buffer systems.