Assay Report Card

ID: ALA3888313

Type: Binding

Description: HCMV Polymerase LANCE TR-FRET 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, 5% DMSO, 235 nM dATP, 350 nM dCTP, 350 nM dGTP, 235 nM dTTP, 12 nM biotin-16-dUTP, 23.5 nM Dig-primer/template, 2 nM GST-UL54. The assay volume is 10 μL. Each reagent is added as follow: 4 μL a+3 μL b+3 μL c; a: compound diluted in compound dilution buffer to obtain 12.5% DMSO; b: enzyme (GST-UL54) in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 25 mM NaCl, 5% Glycerol, 0.67 mg BSA/mL, 1 mM TCEP w/o DMSO (2 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, 783 nM dATP, 1166 nM dCTP, 1166 nM dGTP, 783 nM dTTP, 40 nM biotin-16-dUTP, 78 nM Dig-primer (5'-/Dig/AGC TCG TTT AGT GAA CC-3')/template (5'-GAG GTC AAA ACA GCG TGG ATG GCG TCT CCA GGC GAT CTG ACG GTT CAC TAA ACG AGC T-3') w/o DMSO. The primer and template are annealed in 10 mM Tris-HCl pH 7.5, 50 mM NaCl at a respective concentration of 50 μM. They are incubated at 95 °C. for 5 min in a dry batch block. The block is removed from the dry bath and allowed to cool to RT. Aliquots are made and stored at −20 °C. To perform the assay, 3 μL of the 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. 3 μL of substrate solution is added to each well. The plate is centrifuged at 200×g for 30 sec. Plates are incubated at 37 °C. for 30 min. 5 μL of conjugate solution is added (25 mM Hepes pH 7.5, 0.1 M NaCl, 0.25% Tween-20, 1 mg/mL BSA, 12 mM EDTA, 24 nM Sreptavidin-APC, 342 ng/mL Anti-Dig-Europium). The plates are incubated at RT for at least 120 min. The signal is read on the Envision plate reader (Perkin-Elmer) or equivalent.