| Assay Method Information | |
| | DNMT1 Inhibition Assay |
| Description: | The DNMT1 assays were carried to determine the IC50 with transition state analogs. The reaction (100 μL) containing 50 mM Tris-HCl pH 8.0, 100 mM KCl, 50 ug/mL BSA, 2 mM DTT, 1 μM freshly prepared [Me-3H]-SAM, 1 μM 26 bp hemimethylated DNA substrate and varying concentrations of each inhibitor (0-100 1 μM), incubated at 37° C. with 100 nM DNMT1. The reaction fractions (50 μL) were taken at 2 hours. The methylated DNA was purified by using Micro Bio-spin P-30 columns (Bio-Rad). Purified samples were mixed with 10 mL of scintillation liquid (PerkinElmer), and counted for radioactivity in a Tricarb 2910 TR scintillation counter (PerkinElmer). The values obtained in the absence of DNMT1 in the reaction were used as controls. The assay data were analyzed using GraphPad Prism for IC50 calculations. |
| Affinity data for this assay | |
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