IRDye QC-1 Quencher is the first non-fluorescent quencher that can efficiently quench fluorescence from a wide range of fluorophores from the visible to near-infrared regions (from approximately 500 to 800 nm) in a fluorescence resonance energy transfer (FRET) system. For the first time, you do not need to carefully match the donor’s fluorescence spectrum and the acceptor’s absorption spectrum to determine which acceptor is suitable for your FRET application(s). Just pair the IRDye QC-1 with the visible or near-infrared fluorophore within the wide wavelength range of the quencher.
IRDye QC-1 is a water-soluble, monoreactive NHS ester dye, which allows it to be used to label peptides, proteins, nucleic acids, etc., through the amine group on such molecules. A water-soluble quencher simplifies the labeling and purification process. Protease substrates using the IRDye QC-1 do not require an organic co-solvent in assay buffer systems.
IRDye QC-1 is suitable for FRET-based applications such as protease assays.IRDye QC-1 was used to develop LI-COR’s IRDye HIV-1 Protease Assay Kit and IRDye Beta-Secretase Assay Kit.
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Application Example: IRDye® HIV-1 Protease Assay
The IRDye HIV-1 Protease Assay, as outlined in Figure 1, is based on a highly quenched IRDye 800CW / IRDye QC-1 NIR-FRET peptide substrate.
Figure 1. The principle of the IRDye HIV-1 Protease Assay.
Figure 2. Measurement of HIV-1 protease activity (error bars are ± standard deviation). HIV-1 Protease peptide substrate (20 μL/well at final concentration of 1.0 μM) was titrated with 20 μL/well of recombinant HIV-1 protease from final concentrations of 0.312 nM to 320 nM. The plate was incubated at 37 °C for 1 hour. The reactions were then stopped with 200 μL/well of HIV-1 protease stop solution. The fluorescence intensity was measured on an Aerius® Automated Infrared Imaging System (LI-COR Biosciences).
Figure 3. Inhibition of HIV-1 protease by a characterized inhibitor, Pepstatin A, in a 96-well plate. The serially diluted inhibitor solutions (10 μL/well at final concentrations from 4.11 to 9000 nM) were mixed with HIV-1 protease (10 μL/well at final concentration of 20 nM) for 15 minutes before adding substrate (20 μL/well at final concentration of 1.0 μM). After incubating at 37 °C for 1 hour, the reactions were stopped with 200 μL/well of HIV-1 protease stop solution. The fluorescence intensity was measured on an Aerius Automated Infrared Imaging System.