Assay Report Card

ID: ALA3888314

Type: Binding

Description: HCMV Polymerase Scintillation Proximity Assay: The assay conditions are the following: 10 mM HEPES pH 7.5, 25 mM KCl, 7.5 mM NaCl, 5 mM MgCl2, 0.2 mg BSA/mL, 1 mM TCEP, 1.5% glycerol, 2 μM dTTP, 90 nM 3H-dTTP (minor variations in concentration are possible due to specific activity of stock), 26 nM Poly(dA)/190 nM BioTEG-dT19; 5% DMSO. The assay volume is 30 μL. Each reagent is added at a 3× conc.: 10 μL a+10 μL b+10 μL c; a: compound diluted in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM TCEP with 15% DMSO; b: enzyme (GST-UL54) in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 22.5 mM NaCl, 4.5% Glycerol, 0.6 mg BSA/mL, 1 mM TCEP w/o DMSO (1 nM GST-UL54 is present in the assay); c: substrate in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM TCEP, 6 μM dTTP, 270 nM 3H-dTTP, 78 nM Poly(dA)/570 nM BioTEG-dT19 w/o DMSO. To perform the assay, 10 μL enzyme solution is added to columns 2-12 and 14-24. The enzyme is substituted by the blank solution (b solution without enzyme) for columns 1 and 13 (blanks). The plate is centrifuged at 200×g for 30 sec. 10 μL of substrate solution is added to each well. The plate is centrifuged at 200×g for 30 sec. The plates are incubated at 37 °C. for 40 min. To stop the reaction, 25 μL of SPA beads (5 mg/mL in 0.5 M EDTA) are added and mixed by pipetting up and down. The plates are incubated at RT for at least 15 min. 35 μL of 5 M CsCl is added to the bottom of each well. TopSeal is applied to the plate and the plate is incubated at RT for at least 90 min prior to reading. The signal is read on TopCount plate reader (Perkin-Elmer) or equivalent.