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Assay Method Information

Assay Name:  Radioligand Binding Assay
Description:  IC50 values were determined for compounds A-395 and A-395N at 75 nM of EZH2 trimeric complex (EZH2-EED-SUZ12), 3 μM of human nucleosome and 1 μM of [3H]SAM. 30 μl of reaction mixtures were incubated at 23 C for 70 min. To stop the enzymatic reactions, 150 μl of 10% trichloroacetic acetic acid (TCA) was added, mixed and transferred to filter plates (Millipore; cat. # MSFBN6B10). Plates were centrifuged at 2,000 r.p.m. (Allegra X-15R - Beckman Coulter, Inc.) for 2 min followed by two additional 10% TCA washes and one ethanol wash followed by centrifugation. Plates were dried and 30 μl MicroO (MicroScint-O; Cat. # 6013611, PerkinElmer) was added to each well, and the c.p.m. were measured using Topcount plate reader (PerkinElmer).
Affinity data for this assay
 

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Last update November 1, 2007
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