HIV-1 Integrase Gel-Based Assay

HIV-1 IN gel-based assays were carried out as previously published.^{33 (link)} Briefly, drugs or an equivalent volume of 100% DMSO (dimethyl sulfoxide, used as the drug solvent) was added to a reaction mixture containing 20 nM ³²P-labeled DNA substrate and 400 nM IN in 50 mM MOPS, pH 7.2, 7.5 mM MgCl₂, and 14 mM 2-mercaptoethanol. Reactions were performed at 37 °C for 1 h and stopped by the addition of an equal volume of loading buffer [formamide containing 1% SDS (sodium dodecyl sulfate), 0.25% bromophenol blue, and xylene cyanol]. Products were separated in 16% polyacrylamide denaturing sequencing gels. Dried gels were visualized using a Typhoon 8600 (GE Healthcare). Densitometric analyses were performed using the ImageQuant 5.1 software from GE Healthcare. Data analyses (linear regression, IC₅₀ determination, and standard deviation) were performed using Prism 5.0c software from GraphPad.