Disulfide AMCA-HPDP reactions

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. 

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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See also in sourсe #XX -- [ ]

See also in sourсe #XX -- [ ]

See also in sourсe #XX -- [ ]

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