Labeling Antibodies Using a Maleimido Dye

Fluorophore-maleimide derivatives are effective for labeling sulfhydryl-containing molecules. Maleimide groups react with free thiols at pH 6.5–7.5 forming a covalent bond. Reducing agents should be avoided during the conjugation step. This protocol uses the cross-linker N -succinimidyl S -acetylthioacetate (SATA) to introduce thiol groups on the antibody while maintaining the divalent nature of the antibody. Alternatively, the antibody can be digested and reduced to monovalent Fab fragments, which can then be labeled directly with maleimido derivatives.