Proteins labeled with AMCA

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

The following protocol is a suggested method for labeling a protein with AMCA-HPDP. It is assumed that the presence of a sulfhydryl on the protein has been documented or created. The reaction conditions can be carried out in a variety of buffers between pH 6 and 9. Avoid the presence of extraneous sulfhydryl-containing compounds (such as disulfide reductants) that will compete in the reaction. The inclusion of EDTA in the modification buffer prevents metal-catalyzed sulfhydryl oxidation. Optimization for a particular labeling experiment should be done to obtain the best level of fluorophore incorporation. 

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