| Assay Method Information | |
| | WRN ATPase Assay |
| Description: | ADP-Glo Assay (ADP-Glo™ Kinase Assay from Promega, 10000 assays, #V9102). The assay buffer was comprised of 30 mM HEPES pH7.4 (Gibco, 15630-080), 40 mM KCl (Sigma, P9541-500G), 5% Glycerol (Sigma, G7757-1L), 8 mM MgCl2 (Invitrogen, AM9530G), 0.1 mg/mL BSA (PerkinElmer, CR84-100). Compounds dissolved in DMSO (Sigma, D8418) were plated into a 96-well intermediate plate (BioFil, VWP-032-096) for 3-fold serial titration for 10 points using DMSO, and well-to-well transfer the titrated compounds into another 96-well intermediate plate including assay buffer in advance. Shake the 96-well intermediate plate at 600 rpm for 10 min to fully mix. Specifically, a 4× working mixture of WRN was diluted to a concentration of 8 nM in assay buffer. A 2× working mixture of FORKF (Joshua A. S., Tomasz K., etc., 2019) DNA and ATP was diluted to a concentration of 30 nM and 120 μM, respectively. 2.5 μL titrated compounds from 96-well intermediate plate and 2.5 μL 4×WRN working mixture were delivered into 384-well assay plate (PerkinElmer, 6007290) using electronic pipettor. After centrifuging at 1000 rpm and shaking at 500 rpm, the assay plate was pre-incubated for 30 min. Then, 5 μL 2×FORKF DNA and ATP working mixture was dispensed into assay plate. Plate was sealed, centrifuged and incubated for an additional 40 min. at 37° C. The reaction was stopped with the addition of 10 μL of the first ADP-Glo reagent and incubated for 40 min. to remove the excess amount of ATP. Afterwards, 20 μL of ATP detection reagent was added and incubated for 30 min. before reading. Luminescence output was recorded using BMG CLARIO star plus reader. |
| Affinity data for this assay | |
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