AMCA-HPDP

AMCA-HPDP is N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 -(2 -pyridyldithio) propionamide. It is formed from AMCA plus a 1,6-diaminohexyl spacer off the carboxylate that has been additionally modified at its terminal end with SPDP (Chapter 5, Section 1.1). 

Figure 9.24 AMCA-HPDP reacts with sulfhydryl groups through its pyridyl disulfide end to form reversible disulfide bonds.

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. 

Dissolve AMCA-HPDP in DMSO at a concentration of 0.5 mg/ml. Protect from light. 

In subdued lighting conditions, add 50-100pi of the AMCA-HPDP stock solution to each ml of sulfhydryl-containing protein solution. Mix. 

A general protocol for the use of DCIA for fluorescently labeling proteins that contain sulfhydryl residues may be obtained by following the method discussed for AMCA-HPDP (previous section). After purification of the labeled protein, the F/P ratio of fluorophore incorporation may be determined by measuring its 382nm/280nm absorbance ratio. 

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

AMCA-HPDP is JV-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 -(2 -pyridyldithio)propionamide. It is formed from AMCA plus a 1,6-diaminohexyl spacer off the carboxylate that has been additionally modified at its other end with SPDP (Chapter 5, Section 1.1). The result is a long spacer arm terminating in a pyridyl disulfide group reactive toward free sulfhydryl residues. The reaction of this group with a thiol creates a disulfide bond between the AMCA fluorophore and the molecule being modified. Thus, the fluorescent tag can be specifically cleaved by reduction with DTT or other disulfide reducing agents (Fig. 222). 

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