| Assay Method Information | |
| | Biological Assay |
| Description: | PARG Enzymatic Activity Assay HTRF assay was used to measure the ability of compounds to inhibit the activity of PARG in vitro. C-terminal His6-tag PARG expressed in E. coli was purified and stored at −80° C. in aliquots. Assay measurements were performed with 1×buffer comprising 50 mM Tris pH 7.4, 0.1 mg/mL BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 50 mM KCl and 0.01% Tween 20. Compounds dissolved in DMSO were plated into a 384-well assay plate (PerkinElmer, Catalog #: 6008280) in duplicate using a dispenser (Labcyte, Echo 665), and tested on a 10-point 4-fold serial dilution. Add 5 L Enzyme mix [hPARG (C-terminal His6-tag, 4.2 μM), 65 pM final] to the test wells. The plates were covered and left to incubate for 60 minutes at RT before the addition of 5 L substrate mix [biotinylated-NAD ribosylated PARP1 (6.1 μM), 8 nM final] to initiate reaction. After incubating 10 minutes at RT, add 2.5 L Streptavidin-Eu cryptate (Cisbio, Catalog #: 610SAKLA) and 2.5 L Mab anti 6HIS-XL665 (Cisbio, Catalog #: 61HISXLA) to the plate, and incubate for 60 minutes at RT. Read on a multimode plate reader (PerkinElmer, Envision 2015) in time-resolved fluorescence (TRF) mode, with excitation at 337 nm and emission at both 620 nm and 665 nm. Average HTRF signal of high control (Wells with 1% DMSO) was calculated and as Vehicle Control (VC) |
| Affinity data for this assay | |
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