Use of CyDye fluors for improved FRET protease assays on a BMG LABTECH fluorescence microplate reader
Metalloproteases (MMP) catalyze cleavage of proteins and thereby contribute to tissue remodeling and repair as well as to pathologic events such as tumor invasion. Their activity can be detected with synthetic substrates that are coupled to a fluorophore at one end and to a quencher on the other end. In their native state no fluorescence will occur, as the substrate ends, and accordingly fluorophore and quencher, are found in proximity. Only upon cleavage by MMPs, fluorophore and quencher get separated leading to an increase in fluorescence intensity.
Here, three different peptides were tested: One coupled to the fluorophore Mca and to the quencher DpA, the other two coupled to the fluorophore Cy3B and to the Cy5Q Quencher. Detection of the MMP-2-induced increase in fluorescence intensity with a BMG LABTECH plate reader revealed that the combination of Cy3B:Cy5Q results in higher assay windows compared to the Mca:DpA peptide.