Hydrophobic adsorption chromatography to reduce nonspecific staining by rhodamine-labeled antibodies

Rhodamine absorbed to protein was removed from rhodamine-conjugated antibody solutions by adsorption to hydrophobic macroporous beads (Bio-Beads SM-2) following gel permeation chromatography. This simple technique eliminated the contaminating free dye more effectively than gel filtration alone, but neither significantly reduced the level of fluorescently conjugated antibody nor altered the latter's binding characteristics. Passage of the fluorescent antibody solution over the SM-2 beads eliminated the high background of nonspecific staining caused by internalization of residual free dye.