Amine-Reactive Coumarin Derivatives

Three main forms of amine-reactive AMCA probes are commonly available. One of them is simply the free acid form of AMCA, which can be used to couple to amine-containing molecules using the carbodiimide reaction (Chapter 3, Section 1.1). The other two are active-ester derivatives of AMCA—the water-insoluble NHS ester and the water-soluble sulfo-NHS ester forms—both of which spontaneously react with amines to create stable amide linkages. All of them react under mild conditions with primary amines in proteins and other molecules to form highly fluorescent derivatives. 

AMCA-Sulfo-NHS Sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid MW 431 

AMCA-sulfo-NHS is an analog of AMCA-NHS which contains a sulfonate group on its NHS ring (Thermo Fisher). The negative charge of this group provides enough polarity to 

Dissolve the protein to be modified in 50mM sodium borate, pH 8.5, at a concentration of lOmg/ml. Other buffers may be used for an NHS ester reaction, including 0.1M sodium phosphate, pH 7.5 (Chapter 2, Section 1.4). 

Dissolve AMCA-NHS (Thermo Fisher) in DMSO at a concentration of 2.6mg/ml. Protect from light. 

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DCIA is 7-diethylamino-3-[(4 -(iodoacetyl)amino)phenyl]-4-methylcoumarin, a derivative of the basic aminomethylcoumarin structure that contains a sulfhydryl-reactive iodoacetyl group and a diethyl substitution on its amine. This particular coumarin derivative is among the most fluorescent UV-excitable iodoacetamide probes available (Sippel, 1981) (Invitrogen). 

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