| Assay Method Information | |
| | Assay on Binding Activities of Compounds of the Present Disclosure for PARP1 and PARP2 |
| Description: | In vitro binding activities for PARP1 and PARP2 were tested by the following method.I. Materials and instruments1. PARP1 recombinant protein (Sino Biological, Cat. #11040-H08B);2. PARP2 recombinant protein (BPS, Cat. #80502);3. Fluorescent probe (made in-house using a compound with Cat. #1380359-84-1, Shanghai Hengrui);4. 384-well plate (Corning, 3575);5. Microplate reader PHERAstar FS (BMG Labtech).II. Experimental proceduresTo each well of a 384-well plate was added 8 μL of binding buffer. A fluorescent probe was dissolved in dimethyl sulfoxide, the mixture was diluted to the corresponding concentration, and then the fluorescent probe formulated in dimethyl sulfoxide was 20-fold diluted with the binding buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, and 0.01% IGEPAL) at 2 μL/well. The test compounds were dissolved in dimethyl sulfoxide and diluted to gradient concentrations as required for the experiment, and the compounds at various concentrations formulated in dimethyl sulfoxide were 20-fold diluted with the binding buffer at 2 μL/well. A PARP1 or PARP2 protein was diluted to the corresponding concentration with the binding buffer, and added to a black 384-well plate at 8 μL/well. The plate was incubated at 25° C. for 40 min after the mixture was uniformly mixed. The signal values were read with the FP program in a microplate reader PHERAstar FS. Data were processed using GraphPad software.In vitro binding activities for PARP1 and PARP2 were tested by the following method.I. Materials and instruments1. PARP1 recombinant protein (Sino Biological, Cat. #11040-H08B);2. PARP2 recombinant protein (BPS, Cat. #80502);3. Fluorescent probe (made in-house using a compound with Cat. #1380359-84-1, Shanghai Hengrui);4. 384-well plate (Corning, 3575);5. Microplate reader PHERAstar FS (BMG Labtech).II. Experimental proceduresTo each well of a 384-well plate was added 8 μL of binding buffer. A fluorescent probe was dissolved in dimethyl sulfoxide, the mixture was diluted to the corresponding concentration, and then the fluorescent probe formulated in dimethyl sulfoxide was 20-fold diluted with the binding buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, and 0.01% IGEPAL) at 2 μL/well. The test compounds were dissolved in dimethyl sulfoxide and diluted to gradient concentrations as required for the experiment, and the compounds at various concentrations formulated in dimethyl sulfoxide were 20-fold diluted with the binding buffer at 2 μL/well. A PARP1 or PARP2 protein was diluted to the corresponding concentration with the binding buffer, and added to a black 384-well plate at 8 μL/well. The plate was incubated at 25° C. for 40 min after the mixture was uniformly mixed. The signal values were read with the FP program in a microplate reader PHERAstar FS. Data were processed using GraphPad software. |
| Affinity data for this assay | |
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