| Assay Method Information | |
| | Homogeneous Time Resolved Fluorescence (HTRF) Assay |
| Description: | (1) Each compound to be tested was prepared using gradient dilution method with DMSO and water to obtain a solution with the concentration of 50 nM, 10 nM, 2 nM, 0.4 nM, and 0.08 nM. The concentration of DMSO in the solution of each compound to be tested was 2%.(2) PARP7 enzyme (Cell Chemical Biology 27, 877-887, Jul. 16, 2020; the fusion tags was N-His6-TEV-AviMHHHHHHSSGVDLGTENLYFQSNAGLNDIFEAQKIEWHE) was dissolved in the buffer solution (the pH of the buffer solution was 7.4, and the buffer solution contained 25 mM HEPES (N-(2-hydroxyethyl) piperazine-N′-2-sulfonic acid), 120 mM NaCl, 5 mM MgCl2, 2 mM DTT (Dithiothreitol), 0.002% (ml/ml) Tween-20, 0.1% (ml/ml) BSA (bovine serum albumin) and water) to obtain a PARP7 enzyme solution with the concentration of 6 nM.(3) The RBN011147 (Cell Chemical Biology 27, 877-887, Jul. 16, 2020), MAb Anti His-T b cryptate Gold (Cisbio, Cat. No 61GSTTLF, Lot. No 09A), and Streptavidin-d2 (Cisbio, Cat. No 610SADLF, Lot. No 19G) were diluted with buffer solution (the pH of the buffer solution was 7.4, and the buffer solution contained 25 mM HEPES (N-(2-hydroxyethyl) piperazine-N′-2-sulfonic acid), 120 mM NaCl, 5 mM MgCl2, 2 mM DTT (Dithiothreitol), 0.002% (ml/ml) Tween-20, 0.1% (ml/ml) BSA (bovine serum albumin) and water) to obtain the solution containing fluorophore with the concentration of 10 nM, 0.7 nM, and 2.5 nM respectively. The MAb Anti His-Tb cryptate Gold was the donor fluorophore, and the Streptavidin-d2 was the acceptor fluorophore.(4) 2.5 μl of the solution of the compound to be tested was transferred into 384-well plate, 2.5 μl of the PARP7 enzyme solution was added. The resulting solution was incubated for 15 mins, and then 5 μl of the solution containing fluorophore was added. The resulting mixture was incubated at 25° C. for 3 hrs to obtain the final solution to be tested.(5) The fluorescence signal was read on SPARK plate reader (Tecan), the wavelength of the excitation spectrum of the SPARK plate reader was 320 nm, and the wavelength of the emission spectrum of the SPARK plate reader was 620 nm and 665 nm. The ratio of absorbance at 620 nm to absorbance at 665 nm was calculated for the solution in each well. The ratio was calculated according to the following formula: Ratio=absorbance at 665 nm/absorbance at 620 nm×104.(6) The activation of the compounds to be tested was calculated according to the following formula: Activation (%)=100×(ratiocompound−rationegative)/(ratiopositive−rationegative). Inhibition (%)=100−Activation (%). |
| Affinity data for this assay | |
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