Spectramax paradigm muti mode detection platform

In total, 10 μL final concentration of 50 μM, 20 μM of GC376, and Boceprevir in 25 mM Tris buffer (pH = 8.0) was mixed with 10 μL of 100 μM peptide substrate (Dabcyl-KATVRLQAGNATEE-Edans) solution (Genscript) in a black 96-well plate (Greiner). In all, 30 μL of 1 μM α-Chymotrypsin from bovine pancreas (Solarbio, C8660) in 25 mM Tris buffer (pH = 8.0) was then add to the plate. The RFU value was measured with an excitation wavelength of 360 nm and emission wavelength of 490 nm at 37 °C for 1 h by using SpectraMax Paradigm Muti-Mode Detection Platform (Molecular Devices, USA). Experiments were performed in triplicate. The progress curve of peptide hydrolysis was plotted by GraphPad Prism 8.0.

In all, 10 μL of 100 μM substrate solution (Dabcyl-TSAVLQ↓SGFRKMK-Edans) (Genscript) was added to black 96-well plate (Greiner) with 40 μL final concentration of 200 nM GM^pro or native M^pro in 25 mM Tris buffer (pH = 8.0). The relative fluorescence units (RFU) value was measured with an excitation wavelength of 360 nm and emission wavelength of 490 nm at 37 °C for 1 h by using SpectraMax Paradigm Muti-Mode Detection Platform (Molecular Devices, USA)^{31 (link)}. Experiments were performed in triplicate. Then the progress curve of peptide hydrolysis was plotted by GraphPad Prism 8.0. First 1000 s change of fluorescence value was used to calculate the initial rate v₀ by SoftMax Pro 7.1.