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The Thermo Scientific Pierce FITC and NHS-Fluorescein Labeling Kits contain all the necessary components for labeling antibodies or other proteins and subsequent excess dye removal. Fluorescein isothiocyanate (FITC) crosslinks to amino, sulfhydryl, imidazoyl, tyrosyl and carbonyl groups on proteins; however, only derivatives of primary and secondary amines yield stable products. Reactions are most efficient at pH 8-9 and must be performed in an amine-free buffer such as borate or carbonate/bicarbonate. Generally, the protein is reacted with a 15- to 20-fold molar excess of FITC, which results in several FITC molecules conjugated to each protein molecule.
The purification resin and spin columns eliminate equilibration steps and the need to collect and monitor gravity-flow fractions. This system enables efficient removal of excess FITC and, therefore, accurate determination of the dye-to-protein ratio and exceptional protein recovery.
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