| Assay Method Information | |
| | Biochemical Activity Assay |
| Description: | Biochemical activity of DNMT1 analyzed using SPA technology. Plates (Griener 784075) were pre-stamped with 100 nL/well of compound (11-point, 3-fold serial dilution). Reaction was initiated upon the addition of 5 μL of 2× enzyme mix to wells containing 5 μL of 2× substrate mix. Low control wells contained 5 μL of buffer instead of enzyme. Following a 40 minute incubation, the reaction was quenched upon the addition of 10 μL of stop mixture containing 1 mM SAM (Sigma A7007) and 2 mg/mL PEI beads (Perkin Elmer RPNQ0098). Plates were sealed, centrifuged for 1 min and then read on a Viewlux (Perkin Elmer) using the 613 nm emission filter/300 s read time following a 30 minute dark adapt. Assay conditions prior to quench consisted of 40 nM DNMT1 (601-1600, produced internally), 100 nM 3H-SAM (American Radiolabeled Chemicals ART 0288), 900 nM SAM (New England BioLabs B9003S) and 200 nM hemi methylated DNA oligonucleotide (synthesized at Integrated DNA Technologies, 5′-CCTCTTCTAACTGCCATSGATCCTGATAGCAGGTGCATGC-3′) in 50 mM HEPES (pH 8.0), 2 mM MgCl2, 1 mM DTT, 0.01% NP-40 and 0.01% BSA. |
| Affinity data for this assay | |
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