Common tags used in antibody labeling


Avidin is a molecule forming one of the strongest non-covalent interaction with its ligand, Biotin. The Biotin-Avidin bond formation is very rapid and is very stable independently of the conditions (pH, temperature and other denaturing conditions). The small size of biotin is a main advantage for antibody labeling as it doesn’t affect the properties of the labeled antibody. This small size also confers to biotin the capacity to increase the sensitivity of an assay (as one avidin molecule can bind to 3 labeled biotin molecules).

Biotin labeled antibodies are used in various techniques such as ELISA, Western blot, Immunohistochemistry, FACS…
Several methods exist to make biotin labeled antibodies however, primary amine biotinylation remains the most common technique. Briefly, this chemical reaction results in the labeling of the antibody on the ε-amino groups of the chemically accessible lysines through the use of a N-hydroxysuccinimide ester (NHS) of a biotin analog.

Horseradish Peroxidase (HRP)

HRP is an enzyme reporter commonly used for antibody labeling for various applications such as Western Blot, ELISA or IHC. This enzyme is characterized by a high turnover rate allowing generating a strong signal in a short time period.

3 detection methods are commonly using HRP labeled antibodies:

  • Colorimetric detection: in presence of hydrogen peroxide, HRP catalyzes to reduction of H2O2 into water while oxidizing a substrate such as 4-chloro-1-naphtol (4-CN) or 3,3′,5,5′-Tetramethylbenzidine (TMB) or polymerizing 3′-diaminobenzidine (DAB). These reactions all induce the formation a easily detectable colored precipitate.
  • Chemiluminescence detection: again, in presence of hydrogen peroxide, HRP oxidizes luminol into an unstable excited state intermediate leading to the formation of 3′-aminophtalate and to the emission of light at 425nm.
  • Fluorescent detection is also possible depending on the substrate used.

As biotin, HRP can be conjugated to the e-amino groups of the chemically accessible lysines. It is also possible to link HRP to cysteine residues thanks to a two-step reaction involving successively the reduction of the disulfide bonds of the antibody and the electrophilic addition of a thiol-reactive compound (e.g. a labeled maleimide).

Fluorescein Isothiocyanate (FITC)

FITC is an isothiocyanate derivative of fluorescein rendering it reactive towards primary amines and thiol groups present in the lysine and cysteine residues (when reduced) of biomolecules, respectively. Usually, 3 to 6 fluorescein molecules are conjugated per antibody in order to prevent eventual quenching and solubility issues.
Fluorescent dyes such as fluorescein are commonly used in various lab applications such as Western Blot, flow cytometry and immunofluorescence.