| Assay Method Information | |
| | Polθ Polymerase Activity Assay |
| Description: | The ability of compounds to inhibit Pol 0 polymerase activity was determined by the PicoGreen method in vitro.The His-TEV-SUMO-tagged Polθ protein (amino acids 1792-2590) expressed in E. coli was purified and stored in aliquots at −80° C.The composition of the assay buffer is 25 mM Tris HCl pH 7.5, 12.5 mM NaCl, 0.5 mM MgCl2, 5% glycerol, 0.01% Triton X-100, 0.01% BSA, and 1 mM DTT.Test compounds were diluted in 100% DMSO and diluted three-fold into 10 concentration points in a dilution plate (Greiner-781280) using Bravo (Agilent) according to concentration requirements. The compounds diluted in DMSO were then diluted 20-fold in the assay buffer using Bravo. Further, 2 μl of diluted compounds were transferred to the assay plate (Corning-4512) using Bravo. The purified Pol 0 enzyme and primers ((primer strand: 5′-GCGGCTGTCATAAG-3′, SEQ ID NO: 1):(template strand: 5′-GCTACATTGACAATGGCATCAAATCTCAGATTGCGTCTTATGACAGCCGCG-3′, SEQ ID NO: 2)=1:1.1) were prepared at a working concentration of 2.5× (1.5 nM Polθ and 50 nM PTD) in the assay buffer, transferred to the assay plate at 4 μl per well using an E1-CLIPTIP 12-channel pipette (Thermo, 1-30 l), and incubated at room temperature for 30 min. dNTP (Sigma-D7295) was diluted with the assay buffer to a working concentration of 2.5× (40 uM dNTP), transferred to the assay plate at 4 μl per well (final DMSO concentration of 1%) using an El-CLIPTIP 12-channel pipette (Thermo, 1-30 l), and incubated at room temperature for 60 min. A mixture containing 10 mM EDTA, 25 mM Tris pH 7.5, and a 1:200 diluted PicoGreen dye (Invitrogen-P7581) was added to the assay plate at 6 μl per well to stop the reaction. After 30 min of reaction at room temperature in the dark, fluorescence values were read on EnVision 2105 (PerkinElmer) using the Ex480 nm Em520 nm program, and the raw data was analyzed using XLfit to generate IC50 values. |
| Affinity data for this assay | |
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