Fig. 2.

HTRF assay validation using SARS-nsp14. A) Time course analysis of SARS-CoV nsp14 MTase activity by HTRF assay: 0 or 5 nM of nsp14 (empty or full triangles, respectively) are incubated with 8 μM GpppA and 2 μM SAM at 30 °C for increasing time periods. The reaction by-product (SAH) was quantified by measuring the ratio of emission and excitation fluorescence at 665 and 620 nm, on a PolarStar reader as described in materials and methods. B) Sinefungin IC_{50 (HTRF)} determination. Increasing concentrations of sinefungin (0–0.5 μM) were incubated with 5 nM nsp14, 2 μM SAM, 8 μM GpppA at 30 °C during 20 min and the SAH by-product was detected as described in panel A. The IC_{50 (HTRF)} was then calculated using graphPad Prism equation (n = 3; Mean value ± SD).