| Assay Method Information | |
| | MLL1-WRAD2 Enzyme Assay |
| Description: | Compound potency was assessed through incorporation of 3H-SAM into oligonucleosomes purified from HeLa cells. Specifically, recombinant human MLL1 (aa 3745-3969, GenBank Accession No. NM_005933), WDR5 (aa 22-334, GenBank Accession No. NM 017588), RbBP5 (aa 1-538, GenBank Accession No. NM_005057), Ash2L (aa 2-534, GenBank Accession No. NM_001105214), and DPY-30 (aa 1-99, GenBank Accession No. NM_0325742), all with N-terminal His tag, were expressed in E. coli and mixed at a molar ratio of 1:1:1:1:2. 10 nM of the assembled MLL1-WRAD2 complex was mixed with 100 nM WRAD2 to enhance complex formation before incubation with 0.05 mg/ml nucleosome substrate and compounds (as 10 point duplicate dose response titrations) for 15 min in a buffer consisting of 50 mM Tris (pH 8.5), 5 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.01% Brij-35, and 1% DMSO. Reaction was initiated with 1 μM 3H-SAM and incubated for 1 hour at 30° C. Reaction mixture was transferred to P81 filter-paper and washed with PBS before detection. |
| Affinity data for this assay | |
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