Conjugation of Fluorochromes to Antibodies

Abstract

Immunolocalization of antigen via fluorescence requires that fluorochromes be linked either to the primary antibody (direct method) or to a second antibody (indirect method) to provide a fluorescent signal to mark the site of antibody-antigen binding. Of these two methods, the indirect technique is generally more useful and practical. Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules. Provided are step-by-step instructions for conjugating isothiocyanate derivates of fluorescein and sulfonyl chloride derivatives of rhodamine to the amine groups of antibodies.