PICOMOLAR ASSAY OF NATIVE PROTEINS BY CAPILLARY ELECTROPHORES IS PRECOLUMN LABELING, SUBMICELLAR SEPARATION, AND LASER-INDUCED FLUORESCENCEDETECTION

We report a method for the assay of proteins at concentrations lower t han 10(-10) M with as little as 200 amol of protein, High sensitivity is accomplished by derivatizing the epsilon-amino group of the protein 's lysine residues with the fluorogenic dye 5-furoylquinoline-3-carbox aldehyde and use of a sheath now cuvette fluorescence detector, Most p roteins have a large number of lysine residues; therefore, a large num ber of fluorescent molecules can be attached to each protein molecule. In general, precolumn labeling improves sensitivity but degrades reso lution due to the inhomogeneity of the reaction products from multiple labeling. However, we demonstrate that, through careful manipulation of the separation and reaction conditions, high sensitivity can be obt ained without excessive loss in separation efficiency. Over 190 000 th eoretical plates are obtained for fluorescently labeled ovalbumin.