| Assay Method Information | |
| | HIV RT Filter Binding IC50 Assay |
| Description: | The RT IC50 filter binding assay utilized a synthetic D19/D78mer annealed primer template complex prepared by incubating 50 μM of 19-mer [5′-G TCC CTG TTC GGG CGC CAC-3′] with 50 μM of 78-mer [3-CGA CCG TCC AGG GAC AAG CCC GCG GTG GCG ATC TCT TGA CAT TCC GTA ACC TTC GTA TTT TAA GCA TCA TAG TAC ACA-5] in 50 mM Tris pH 7.8 at 95° C. for 5 minutes, 55° C. for 15 minutes, and 37° C. for 10 minutes. 22.5 nM HIV RT was added to reaction mix containing 150 mM Tris-Cl pH 7.8, 180 mM KCl, 0.33 mM DTT, 0.9 mg/mL bSA, 12.6% glycerol, 3 uM D19/D78 mer. Serial dilutions of compounds were added at equal volumes and reactions were heated to 37° C. The reactions were initiated with equal volumes of dNTP mix containing 150 μM each of noncompeting dNTP and 1.2 μM of competing dNTP, and 0.1 μCi/μL competing dNTP. After 5 minutes at 37° C., 5 μL reactions were spotted on DE81 paper, washed three times in 0.125M Na2HPO4, dried, and quantified by autoradiography. IC50 values were calculated in Prism by non-linear regression analysis using the dose-response (variable slope) equation (four-parameter logistic equation): Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)). |
| Affinity data for this assay | |
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