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 detect ion by laser-induced fluorescence. NanoOrange binds noncovalently with hydr ophobic protein regions to form highly fluorescent complexes. Kinetic measu rements of complex formation on the microchips suggest that the reaction ra te is near the diffusion limit under the conditions used for protein separa tion. Little or no band broadening is caused by the postcolumn labeling ste p. Lower limits of detection for model proteins, cc-lactalbumin, P-lactoglo bulin A, and beta-lactoglobulin B, were <0.5 pg (similar to 30 amol) of inj ected sample. The relative fluorescence and reaction rates are compared wit h those of a number of other fluorogenic dyes used for protein labeling.