By Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde (auth.), Edwin Yunhao Gong (eds.)
Although antiviral medicines were effectively built for a few viral illnesses, there continues to be a transparent, unmet clinical have to enhance novel antiviral brokers for the keep an eye on and administration of many viruses that at the moment haven't any or constrained healing procedures in addition to a necessity to beat the constraints linked to the present antiviral medications, equivalent to hostile results and emergence of drug-resistant mutations. the second one variation of Antiviral tools and Protocols features:
- All chapters are new and written by means of specialists within the box, reflecting the foremost fresh technical advances in antiviral learn and discovery.
- This variation makes a speciality of many vital human viruses, reminiscent of human immunodeficiency virus kind 1 (HIV-1), hepatitis viruses (hepatitis B and C viruses), herpes viruses, human respiration syncytial virus (RSV), and influenza virus, whereas additionally that includes a few very important rising viruses, equivalent to dengue virus, West Nile virus, and chikungunya virus.
- As a quantity within the hugely profitable Methods in Molecular Biology sequence, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, quite simply reproducible laboratory protocols, and tips about troubleshooting and heading off recognized pitfalls.
Comprehensive and state-of-the-art, Antiviral equipment and Protocols, moment Edition will function a good laboratory reference for pharmaceutical and educational biologists, medicinal chemists, and pharmacologists in addition to for virologists within the box of antiviral examine and drug discovery.
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Purified protein is obtained by performing in succession: affinity chromatography, removal of the HIS6-tag, ion exchange chromatography, and gel filtration chromatography. Finally, the purified LEDGF/p75 is concentrated using an Amicon Ultra-15 filter. 4) supplemented with 10 % glycerol, 5 mM DTT, and 120 mM NaCl. 2 Oligos for the 3 ′-Processing Assay 1. INss (sense strand): 5′-TGTGGAAAATCTCTAGCAGT-3′Alexa Fluor® 488 fluorophore. 2. INas (antisense strand): dabcyl-5′-ACTGCTAGAGATTTTCC ACA-3′. 3 Primers for the STT Assay 1.
Efavirenz, a marketed NNRTI [12, 13] as a reference compound. An SPA-Based HIV-1 Reverse Transcriptase Assay 21 reverse transcriptase, dTTP/[3H]dTTP 4 h incubation at 37°C Streptavidin SPA bead (unstimulated) Streptavidin SPA bead (stimulated) Stop reaction with stop solution. Read-out with TopCount Biotin Fig. 1 Principle of the HIV-1 RT assay using SPA. 2 Consumables 1. F96 MicroWell™ MaxiSorp™ plate (Nunc). 2. TopSeal-A 384, Clear Self-Adhesive Topseal for 384-well Microplates (PerkinElmer).
IC50 is defined as the compound concentration that inhibits 50 % of the HIV-1 RT activity (reduction of CPM) compared with untreated (= without compound) controls. First, the % inhibition is calculated for every concentration using the following formula: % inhibition = ((C+ − X)/ (C+ − C−)) × 100 in which X is the CPM measured at each compound concentration. C+ is the median CPM of the positive control wells and C− is the median CPM of the negative control wells (background). For each compound concentration, the average inhibition of the duplicates is calculated.
Antiviral Methods and Protocols by Peggy Geluykens, Koen Van Acker, Johan Vingerhoets, Christel Van den Eynde (auth.), Edwin Yunhao Gong (eds.)