| Assay Method Information | |
| | PARP2 FP Assay |
| Description: | Compounds were dissolved and 4-fold serially diluted in DMSO. Compounds were transferred by an Echo to the assay plate to reach final concentrations ranging from 10 uM to 0.01 nM. Assay buffer was composed of 50 mM Tris pH 8.0, 10 mM MgCl2, 150 mM NaCl and 0.001% Triton X100. PARP2 (BPS Bioscience, Cat #80502) was diluted in the assay buffer to make 40 nM (2xfinal concentration) enzyme solution. 5 ul of the enzyme solution was then added to each well with compound and the plate was incubated at room temperature for 10 minutes. Probe (TOCRIS, Cat #6461) was diluted in the assay buffer to make 6 nM (2xfinal concentration) probe solution and 5 ul of the probe solution was added to the wells to start the reaction. The plates were incubated for 4 hours at room temperature. Fluorescence polarization assays (FP) were performed to measure the activity of kinase. Fluorescent polarization of the PARP2 sample was measured by exciting samples at 480 nm and detecting emission at 535 nm in both parallel and perpendicular channels on Envision (Perkin Elmer). The percentage inhibition was calculated by using the formula: % Inhibition=100x(mPHC-mPsample)/(mPHC-mPLC). The low and high control values (LC and HC) were generated from wells with only assay buffer or with enzyme and probe treated with 0.1% DMSO, respectively. |
| Affinity data for this assay | |
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