Labeling with biotin-hydrazide

If a free amino group forms a portion of the protein that is essential for activity (e.g., the antigen-combining site for antibody), biotinylation with the succinimide ester will lower or destroy the activity of the protein, and other methods of labeling should be tried. Biotin hydrazide has been used to modify the carbohydrate moieties of antibodies (10,11). Other alternatives are the thiol-reactive biotin maleimide (12) or biotin iodoacetamide (13). 

Other sites on IgG can be used for labelling. The oligosaccharides (which are chiefly in the Fc part of the molecule) can be oxidized and the resulting aldehyde groups reacted with hydrazide derivatives of, for example, biotin (1). 

Figure 8.3. Examples of small molecules employed In site-specific labeling methods. (A) Structures of FLAsH and a 4 -phosphopantetheinyl-fluorophore conjugate. (B) Structure of a BG-fluorophore conjugate and a scheme for the covalent attachment of the fluorophore to AGT. (C) Structures of biotin and the ketone analogue of biotin. Attachment of the ketone analogue to an AP-protein fusion and subsequent reaction with a fluorophore hydrazide for covalent fluorophore attachment.

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