| Assay Method Information | |
| | NS5B RNA Polymerase Assay |
| Description: | NS5B RNA polymerase reactions for IC50 determination were performed in 96-well microtiter plates in 20 μL reactions containing assay buffer (50 mM Na+ HEPES, 1 mM MgCl2, 0.75 mM MnCl2, 2 mM DTT, pH 7.5), 1 U/μL SUPERase. In (Life Technologies), 20 ng/μL IRES RNA template, 1 μM each ATP, CTP, GTP, and UTP (Life Technologies) including [α-32P]-CTP at a final specific activity of 50 Ci/mmol (PerkinElmer), test compounds in 10-point half-log dilution series, and NS5B polymerase. Reactions were incubated at 27° C. for 60 minutes and terminated by dilution to 100 μL in 1× stop solution (final concentrations 144 mM Na+ citrate, 1.44 M NaCl, 10 mM EDTA, pH 7.0). 80 μL stopped samples were applied by vacuum to nylon membrane filtermats (PerkinElmer), washed 4× in 60 mM Na+ citrate, 600 mM NaCl (pH 7.0), rinsed sequentially in H2O and EtOH, dried, and counted in a MicroBeta2 counter with scintillation cassette (PerkinElmer). |
| Affinity data for this assay | |
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