Assay Method Information

Assay Name:  Enzymatic Activity Assay
Description:  Enzymatic activity assay was conducted using EZH2(Y641F) TR-FRET assay KIT from Cisbio company on compounds that were shown to be active in primary screening, wherein GSK126 (purchased from Shanghai Haoyuan Chemexpress Co., Ltd.) and EPZ6438 (CAS No. 1403254-99-8, purchased from Shanghai Haoyuan Chemexpress Co., Ltd.) were used as control.2 μL of PRC2 protein complex that contained 14 ng protein (comprising EZH2(Y641F), EED protein, SUZ12 protein, RbAp48 protein and AEBP2 protein, purchased from BPS Bioscience, USA, catalog #51017) and 4 μL of 10 mM of 3-fold gradient diluted compounds of the invention were mixed respectively, and incubated for 5 minutes in a 384-well shallow plate at room temperature. Then the reaction substrates, 2 μL of 2.5 μM Histone3K27ME1 (purchased from AnaSpec, USA, catalog #65366) and 2 μL 75 μM adenosylmethionine (SAM) (purchased from sigma, USA, catalog # A7007), were mixed and incubated at room temperature for 4 hours. Subsequently 5 μL of the assay antibody, H3K27me3-Eu(K)Ab (purchased from cisbio bioassays, USA, #61 KC3KAE) and 5 μL Streptavidin-XL665 (purchased from cisbio bioassays, USA, #610SAXLA), were added and incubated for 1 hour at room temperature. The resultant was read on Spectramax i3 (Molecular Devices, USA) which was set in a TR-FRET reading mode, to obtain absorption data at wavelength 665 nm and 620 nm.
Affinity data for this assay
 

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