Fluorescein isothiocyanate (FITC) is a fluorochrome dye that absorbs ultraviolet or blue light causing molecules to become excited and emit a visible yellow-green light. FITC is a commonly used marker for antibodies in immunofluorescent techniques since the conjugation of FITC to proteins is relatively easy and does not, in general, destroy the biological activity of the labeled protein. FITC is also widely used as a hapten to label different proteins. It is typically available as a mixture of isomers, fluorescein 5-isothiocyanate (5-FITC) and fluorescein 6-isothiocyanate (6-FITC). FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. CAS number of FITC: 27072-45-3 (mixture); 3326-32-7, 5-isomer; 18861-78-4 (6-isomer). 5-FAM is a single isomer with Ex/Em of 490 nm/520 nm. The single isomer, 6-FAM, contains a carboxylic acid that can be used to react with primary amines via carbodiimide activation of the carboxylic acid. Fluorescein is the most common fluorescent derivatization reagent for labeling biomolecules.
