| Assay Method Information | |
| | RNA-Dependent RNA HCV NS5B (polymerase) Assay and IC50 Determination |
| Description: | The reaction mixtures consisted of 50 mM Hepes-KOH, pH 7.5, 5 mM MgCl2, 5 mM DTT, 2% glycerol, 0.01% Triton X-100, 0.5 uM polyA:U16 substrate, purified HCV RNA-dependent RNA polymerase, 10 μM UTP, and 32P-UTP (Perkin Elmer). The reaction mixtures incubated at 30° C. for 60 minutes, and then filtered through Zeta probe membrane (BioRad). The filter was washed with 5×SSC (75 mM sodium citrate, pH 7 and 750 mM NaCl), and the radiolabeled RNA products were quantitated by microbeta (Perkin Elmer). For IC50 determination, different concentrations of inhibitors were added to the polymerase reaction mixtures, and incubated at 37° C. for 60 minutes. IC50 values were determined using GraFit (Erithaus software). |
| Affinity data for this assay | |
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