| Assay Method Information | |
| | EZH2 Enzyme Activity Assay |
| Description: | The compounds were dissolved in DMSO, formulated into 10 mM solutions and serially diluted with DMSO to obtain 100-fold the final concentration. 200 nL of the solutions of the compounds was transferred to a 384-well plate (Perkin Elmer, Cat. No. 6007299) by using Echo550. EZH2 (BPS, Cat. No. 51004) was diluted with 1×assay buffer (50 mM Tris-HCl 9.0, 0.01% Tween-20, 1 mM DTT) to 2-fold the final concentration (EZH2: 3 nM). A mixed solution of H3K27(21-44) and [3H]-SAM (PerkinElmer, Lot. No. 2146246) (H3K27(21-44): 200 nM, [3H]-SAM: 100 nM) was formulated. The EZH2 diluted solution was taken and added to the 384-well plate at 10 μL/well (10 μL of 1×assay buffer was added in the control group). The plate was sealed and then incubated at room temperature for 15 minutes. 10 μL of the mixed solution of H3K27 (21-44) and [3H]-SAM was added to each well. The plate was sealed and then incubated at room temperature for 60 minutes. 50 μM of SAM (Sigma, Cat. No. A7007) was formulated at low temperature and added to the 384-well plate at 10 μL/well. The sample was taken at 25 μL/well, transferred to a flashplate and incubated at room temperature for at least 1 hour. The flashplate was washed 3 times with a plate washing liquid (dH2O+0.1% Tween-20) and read by using Microbeta. |
| Affinity data for this assay | |
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