| Assay Method Information | |
| | Enzyme-Linked Immunosorbent Assay |
| Description: | EZH2 wild type: Experimental method: avdin of a final concentration of 100 nM was used to coat 96-well plate at 10 μL/well, placed in a wet box and shaken overnight, and then 100 μL 3% BSA per well was added and blocked for 1 h at room temperature. PRC2 complex (EZH2/EED/SUZ12/RbAp48/AEBP2), H3 (21-44) me0 substrate, methyl donor SAM and compound were added into each well of the blocked 96-well plate. The total reaction system was 100 μL, placed in a wet box and allowed to react for 1 h on shaker at room temperature. The plate was washed with TBS-T [20 mM Tris-HCl (pH 7.2-7.4, room temperature), 150 mM NaCl, 0.1% (v/v) Tween-20] for 3 times, blocked with 3% BSA for 10 min, and anti-H3K27me3 antibody was added and incubated for 1 h in a wet box on shaker at room temperature. The plate was washed again with TBS-T for 3 times, and blocked with 3% BSA per well for 10 min. Horseradish peroxidase-labeled secondary antibody was added and reacted in a wet box at room temperature for 1 h, and finally, washed with TBS-T for 3 times. 2 mg/ml OPD color developing solution (1004/well) was added for coloring, and the reaction was stopped with 2M H2SO4 (50 μL/well). The plate was read by a plate reader at 490 nm and IC50 of the compound was calculated using SoftMax Pro 5.4.1 software. |
| Affinity data for this assay | |
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