Fluorescein 5-isothiocyanate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

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Reaction Steps

To 0.34 mg fluorescein isothiocyanate placed as a dry solid in a screw-capped test tube was added 64 micrograms of N,N,N-trimethylammoniumbenzene iodide in 25 microliters of water, 0.2 ml 1M sodium carbonate buffer, pH 9.5, and 17 mg of the antibody product obtained in Example 3 in 2 ml PBS. The reaction was continued for six hours on ice, after which the solution was dialyzed against 0.015M NaCl, 0.01M sodium phosphate. This solution was applied to a DEAE-cellulose column equilibrated in the same buffer. Step-wise increases in NaCl concentration gave fractions eluting between 0.05M NaCl and 0.15M NaCl which were pooled and dialyzed against PBS. The solution was sterilized by passage through a 0.2 micron filter to yield a fluorescein labeled antibody.

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