| Assay Method Information | |
| | Biochemical Activity Assays |
| Description: | WRN helicase core (8xHis-tev-WRN (480-1251)) was expressed in Sf9 insect cells and purified using HiTrap TALON affinity purification followed by heparin column. The pure fractions were pooled and desalted/buffer exchanged into storage buffer using a HiTrap desalting column. WRN activity assays were performed in a multiplex fashion by monitoring both DNA unwinding using a quenched dsDNA substrate (dsDNArev-Cy3: 5’-Cy3-GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT; dsDNAfwd-FQ: 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC-IowaBlackFQ-3’, Integrated DNA Technologies) and ATPase activity using an ADP-Glo Kinase Assay (Promega). First, WRN alone was pre-incubated with DMSO or compound of interest for 15 minutes. To initiate the reaction, ATP, dsDNA substrate, and ssDNA competitor (ssDNA_comp: GAACGAACACATCGGCTAC) were added and incubated at room temperature for 20 minutes at which point DNA unwinding activity was monitored by measuring Cy3 fluorescence. After obtaining the reading, the ATPase activity was measured using the ADP-Glo Kinase Assay following the manufacturers recommended protocol. The final reaction conditions were: 12.5nM WRN, 1mM ATP, 15nM dsDNA substrate, 1.5uM ssDNA competitor in reaction buffer composed of 50mM Tris-HCl pH 8.0, 50mM NaCl, 2mM MgCl2, 0.01% v/v Tween-20, 2.5µg/mL poly(dI-dC), 1mM DTT. Compounds were assayed in a 10-point dose response in duplicate at a 1% DMSO final assay concentration. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |