Fluorescein Isothiocyanate Labeling

Fluorescein isothiocyanate (FITC) has the ability to permanently label biomolecules with a unique detectable property, which can be used to detect or track the conjugate as it interacts with other biomolecules. At Biologics International Corp. (BIC), whether using custom antibody in enzyme-linked immunosorbent assay (ELISA) or western blotting, our FITC labeling service allows for simplified detection. Please contact us for project quotations or any questions.


  • Comprehensive service: A full range of services from protein or antibody production to FITC labeling.
  • Mature technology: With stable and mature platforms, our professional team offers you FITC labeled proteins or antibodies with high bioactivity and sensitivity.
  • Diverse selection: We can offer a wide variety of labels, including horseradish peroxidase (HRP), biotin, and isotopes.


Structure of FITC

FITC, an isothiocyanate derivative of fluorescein, is primarily synthesized through modification of its lower ring at the 5-carbon positions. The isothiocyanate of FITC can react with primary amine groups of proteins or antibodies, and this property is used for labeling. The fluorescent properties of FITC include an absorbance maximum at about 495 nm and an emission wavelength of 520 nm.

Advantages and Applications of FITC Labeling

Among the large number of fluorescent dyes, FITC has been one of the most popular fluorophores due to its high absorptivity, excellent fluorescence quantum yield, and good water solubility. Like other fluorescein derivatives, FITC can produce a detectable signal without the need for additional reagents for detection. This feature makes FITC extremely versatile for detecting protein or antibody location and activation, identifying protein or antibody complex formation and conformational changes, and monitoring biological processes in vivo.

With these advantages, FITC has been used in numerous applications. FITC labeled antibodies can detect antigens in cells, tissue sections, and western blots. Tagging molecules with FITC is also useful in detecting proteins after electrophoretic separations and for microsequencing analysis of proteins and peptides.

The Protocol of FITC Labeling

  1. Select a correct buffer for the protein or antibody, such as sodium carbonate (0.1M, pH 9.0 or 10.0).
  2. Prepare a protein solution at a high concentration (at least 2 mg/ml). Higher concentration is generally easier for labeling.
  3. Dissolve FITC in dry DMSO at a concentration of 1 mg/ml. This step is preformed in a laboratory in the dark, as it requires protection from light. Caution is needed because the isothiocyanate group is stable in the aqueous solution for only a short period of time and FITC can also break down and lose activity upon storage. Using fresh reagent during the labeling process is the best method.
  4. Add 100 μl of FITC solution to each milliliter of protein solution slowly and mix the complex gently. Derivatization of a protein cannot be carried out at a very high level. Modifications of proteins involve adding with a four to five fluorescein molecules per protein molecule, as it is considered to be optimal.
  5. Stir the complex for at least 2 h at 4 ℃ in the dark.
  6. Purify the derivative by gel filtration using a PBS buffer or another suitable buffer for the particular protein being modified, such as applying the reaction mixture onto a PD10 column previously equilibrated with the desired buffer.
  7. For specific applications, detect the fluorescence generated by the labeled protein or antibody and evaluate the impact of labeling.


Fluorescein (molecular weight is about 332 Da) typically contains several combined aromatic groups or planar or cyclic molecules with several π bonds. Fluorescein derivatives are often generated through modification at the C-5 or C-6 position on the lower aromatic ring. In its most elementary form of fluorescein and its derivatives, the presence of a multi-ring aromatic xanthene core structure creates the phenomenon of re-emitting light upon light excitation.

Principle of Fluorescence

Due to the characteristics of the structure of fluorescent molecules and their derivatives, they can absorb photons of energy at one wavelength (wavelength range is about 488-495 nm) and subsequently emit energy at another wavelength (wavelength generally range between 518 and 525 nm). The quantum energy levels of electrons of some compounds increase with the uptake of photons; this process is known as excitation. The excess energy of an excited fluorophore can be released as photons of light as the electrons return to a lower ground-state energy level, and this process of light emission is the so called fluorescence.

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