| Assay Method Information | |
| | In Vitro Methyltransferase Activity Assay |
| Description: | Recombinant PRC2 (EZH2-Y641F) was purchased from Active motif, S-adenosyl-methionine (SAM) and Poly-L-lysine (PLL) were purchased from Sigma-Aldrich, and H3(1-50)K27me1 peptide was purchased from Cisbio. LANCEUltrasystem (Perkinelmer) was used for detection system. In the enzyme activity test, compounds to be tested were diluted by 8 gradient points in a ratio of 1:3, and added into each well in a reaction plate, and 100 ng recombinase was added, followed by a buffer [20 mM Tris pH8.5, 2 mM MgCl2, 0.01% Tween-20, 1 mM TCEP] containing 2.5 uM SAM/250 nM H3 (1-50)K27me1 premixtures. Enzymatic reaction thus began at room temperature. After reacted for 3 hours, a detection solution premixed with PLL, detection antibody and Ulight was added, and reacted for 1 hour at room temperature. The fluorescence value was then read on a Tecan infinite pro. IC50 was calculated by fitting in a four-factor model in the XLfit software. |
| Affinity data for this assay | |
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