Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.