| Assay Method Information | |
| | HIV-1 RT Inhibitory Assay |
| Description: | The NNRTI compound to be evaluated (NVP) was serially diluted in 50% DMSO. The reaction mixtures containing 150 nM labeled primer−template hybrid, 100 μM dATP, 0.25 or 6 mM free Mg2+ (adjusted according to the concentration of dATP), and 5% DMSO were preincubated at 37 °C for 3 min in a total volume of 8.5 μL of reaction buffer (see above). Twenty-five nM HIV-1 or K103N RT (at similar activities of primer extension) was preincubated with different dilutions of NVP (as noted in the figure legends) for 10 min at room temperature. Reaction was then initiated by adding the NVP/ RT mix to the primer−template hybrid. The final pH of the reactions was 7.7. After extension for 3 min, the reactions were stopped by adding 12.5 μL of 2× gel loading buffer and the samples were resolved in a 16% denaturing polyacrylamide−7 M urea gel. |
| Affinity data for this assay | |
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