| Assay Method Information | |
| | Fluorescence-based Assay |
| Description: | The fluorescence-based assay outlined in Figure 2 a was performed in black, low volume 384-well plates with a fi nal volume of 10 L per well. Nine uL of assay buffer (20 mM Tris-HCl, 100 mM KCl, 0.1% BSA, 0.01% Tween-20, pH 7.5) was added toeach well, followed by the addition of 1 uL of DMSO (control) or 50 M compound in DMSO. Drug addition and subsequent mixing was performed by an automated robotic system (Sciclone ALH3000 Workstation with the low-volume 384 mandrel array and disposable tips, PerkinElmer). A total of 20 nL of 25 uM OGG1 in assay buffer + 0.15% Tween was dispensed into each well via a HP D300 digital dispenser (Tecan). Plates were incubated at RT for 5 10 min and then 20 nL of 12.5 uM 8-oxo-Gua-containing substrate (diluted in assay buffer + 0.15% Tween) was dispensed into each well using the same method. The final concentration of each component in the reaction was 5 uM drug, 50 nM enzyme, and 25 nM substrate. Plates were incubated for 30 min at 37 °C, and TAMRA fluorescence in each well was measured using the Biotek Synergy 4 platereader (Filters = Ex 528/20, Em 600/40. Mirror = Top 570 nm with polarizer). Backgroundsubtracted fluorescence values were calculated for each well, and any compound that had 40% decrease in fluorescence compared with the no inhibitor control was identified as a hit. Control wells containing just substrate or just substrate + enzyme were used to calculate Z values for each plate. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |