| Assay Method Information | |
| | Exonuclease Assay |
| Description: | To evaluate the effect of compounds on TREX1 activity, test compounds were serially diluted (11-point, 3-fold) from 10 mM stock solutions and delivered to 384-well low-volume assay plates in 80 nL DMSO using an acoustic dispenser. Next, 4 μL of human TREX1 (0.5 nM), or murine TREX1 (1 nM), diluted in assay buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100 μg/mL BSA, 0.002% Triton X-100, 2 mM DTT), was added to the assay plate. After incubating for 30 minutes, 4 μL of labeled DNA oligonucleotide (500 nM) in assay buffer was added to initiate TREX1 exonuclease activity. The reaction was allowed to proceed for 45 minutes at room temperature prior to the addition of 4 μL of 150 mM EDTA to halt TREX1 activity. Assay plates were equilibrated for an additional 30 minutes and read on an EnVision Plate Reader (Perkin Elmer) to measure fluorescence emission at 535 nm following excitation at 485 nm. Fluorescence was plotted as a function of log molar compound concentration and fit to four-parameter dose-response equation to determine compound IC50. |
| Affinity data for this assay | |
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