Author: Ross D. Overacker; Somdev Banerjee; George F. Neuhaus; Selena Milicevic Sephton; Alexander Herrmann; James A. Strother; Ruth Brack-Werner; Paul R. Blakemore; Sandra Loesgen
Title: Biological Evaluation of Molecules of the azaBINOL Class as Antiviral Agents: Specific Inhibition of HIV-1 RNase H Activity by 7-Isopropoxy-8-(naphth-1-yl)quinoline Document date: 2019_1_23
ID: m2zw8eq4_52
Snippet: To determine if B#24 was capable of non-specifically inhibiting luciferase activity in the 557 HIV-1 pseudotyped assay, we implemented a cell-free assay with purified recombinant luciferase 558 (Promega) and luciferin (BrightGlo Luciferase Assay System; Promega) following a previous 559 protocol. 38 Protein and reagents were prepared and stored according to manufacturer's 560 instructions. Preliminary experiments established the concentration of .....
Document: To determine if B#24 was capable of non-specifically inhibiting luciferase activity in the 557 HIV-1 pseudotyped assay, we implemented a cell-free assay with purified recombinant luciferase 558 (Promega) and luciferin (BrightGlo Luciferase Assay System; Promega) following a previous 559 protocol. 38 Protein and reagents were prepared and stored according to manufacturer's 560 instructions. Preliminary experiments established the concentration of luciferin to be used in the 561 assay in order to closely mimic the protein signal in single-round infectivity assay and give 562 maximal sensitivity in luminescence readings. Reactions were carried out in white 96 well-plates 563 (Gibco; ThermoFisher Scientific) by combining compounds with 0.5 µg/mL recombinant 564 luciferase in 1x PBS buffer and 1 mg/mL BSA (VWR). Reactions were initiated by the addition 565 of 30 µL luciferin substrate to wells. Luminescence readings were immediately recorded and 566 normalized using appropriate controls with vehicle solvent (1% DMSO). The compounds 567
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