How to label antibodies?

Antibody labeling, or antibody conjugation, is the process of covalently attaching a label (e.g. an enzyme or fluorophore) to a primary or secondary antibody. These labels have the capacity to generate a measurable signal that facilitates in the detection of the antibody-antigen complex. Antibody conjugates are widely used in a broad range of immunological applications including Western blot, ELISA, flow cytometry, immunohistochemistry (IHC), immunocytochemistry (ICC) and immunofluorescence (IF).

Two methods are commonly used to label antibodies. The first, and the simplest method, is to label primary amines (-NH₂) that exist at the N-terminus of each polypeptide chain and in the side-chain of lysine residues of antibodies. This requires the use of fluorophores modified with amine-reactive chemical groups such as succinimidyl esters (SE) and NHS esters. The second method is to label thiol groups (-SH) that are located in the side-chain of cysteine residues. This requires the use of fluorophores modified with thiol-reactive chemical groups such as maleimides.

If conjugation chemistry is not your strong suit, consider using antibody labeling kits such as ReadiLink™ Rapid iFluor® Dye Antibody Labeling Kits. These kits produce fluorescent antibody conjugates in two easy mixing steps with 100% conjugate recovery.