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| | PARG Biochemical Assay |
| Description: | Briefly, PARG in vitro assays were conducted in a total volume of 15 μL in a standard 384-well format. A total of 5 μL of human full length PARG (AstraZeneca), used at a final reaction concentration of 65 pM, was added to 5 μL of Bt-NAD ribosylated PARP1 substrate (AstraZeneca) at a final reaction concentration of 4.8 nM in assay buffer (50 mM Tris pH 7.4, 0.1 mg mL−1 BSA, 3 mM EDTA, 0.4 mM EGTA, 1 mM DTT, 0.01% Tween 20, 50 mM KCl). The reaction was incubated at RT for 10 min, and then 5 μL of detection reagent was added. Detection reagent consists of 42 nM mAb anti-6HIS XL665 (CisBio: 61HISXLB) and2.25 nM streptavidin europium cryptate (CisBio: 610SAKLB), both at 3× working stock concentrations (final concentrations of 14 nM and 0.75 nM, respectively), in detection buffer (50 mM Tris pH 7.4, 0.1 mg mL−1 BSA and 100 mM KF). Following incubation at RT for 60 min in the dark, TR-FRET signal was measured at λEx 340 nm and λEm 665 nm and λEm |
| Affinity data for this assay | |
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