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| | Polymerase Assay |
| Description: | Assay Protocol: Either wild type or S282T (Migliaccio, et al., J. Biol. Chem. 2003, 49164-49170; Klumpp, et al., J. Biol. Chem. 2006, 3793-3799) mutant polymerase enzyme was used in this assay. NS5b polymerase assay (40 uL) was assembled by adding 28 uL polymerase mixture (final concentration: 50 mM Tris-HCl at pH 7.5, 10 mM KCl, 5 mM MgCl2, 1 mM DTT, 10 mM EDTA, 4 ng/uL of RNA template, and 75 nM HCV D21 NS5b polymerase) to assay plates followed by 4 uL of compound dilution. The polymerase and compound were pre-incubated at 35 C. for 10 minute before the addition of 8 uL of nucleotide substrate mixture (33P-gamma-labeled competing nucleotide at KM and 0.5 mM of the remaining three nucleotides). The assay plates were covered and incubated at 35 C. for 90 min. Reactions were then filtered through 96-well DEAE-81 filter plates via vacuum. The filter plates were then washed under vacuum with multiple volumes of 0.125 M NaHPO4, water, and ethanol. |
| Affinity data for this assay | |
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