What is the difference between Fluorescein and FITC? and methods of labeling?
FITC (Fluorescein isothiocyanate) is activated precursor used for the Fluorescein labeling. For the efficient N-terminal labeling, a seven-atom aminohexanoyl spacer (NH2-CH2-CH2-CH2-CH2-CH2-COOH) is inserted between the fluorophore (fluoroscein) and the N-terminus of the peptide. This spacer helps to separate the fluorophore from its point of attachment, potentially reducing the interaction of the fluorophore with the biomolecule to which it is conjugated and making it more accessible to secondary detection reagents.