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|The caspase-3/7 substrate is a bisamide derivative of the fluorophore rhodamine 110 (R110). Peptides are covalently linked to each of the amino groups of rhodamine 110 (R110), thereby suppressing both the visible absorption and fluorescence of the dye. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted in a two-step process, first to the fluorescent monoamide and then to the even more fluorescent R110. The R110 cleavage product has spectral properties similar to those of fluorescein and a large extinction coefficient, thus providing ease of use under standard fluorescein filter set-ups and excellent signal to background ratios. The substrate can be used to continuously measure enzyme activity in cell extracts and purified enzyme preparations using a fluorometer or fluorescence microplate reader.
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