| Assay Method Information | |
| | Radioactivity-Based Assay |
| Description: | In this assay [3H]AdoMet (PerkinElmer Life Sciences; catalog number NET155V250UC)was used as a methyl donor to methylate biotinylated histone peptide substrates. Reaction mixtures contained 10 μM biotinylated peptide and 200 μM AdoMet (4 μM [3H]AdoMet plus 196 μM cold AdoMet) in 10mM Tris-HCl buffer, pH 8.5, with 0.01%Triton X-100 and 10 mM DTT in a final volume of 20 μl. The reaction was started by adding PRDM9(final concentration of 1 nM). Samples were incubated for 30 min at 23 °C and the reaction was quenched by addition of 20 μl of 7.5 M guanidinium hydrochloride followed by addition of 160 μl of 20 mM Tris-HCl buffer, pH 8.0. Samples were added to wells of a streptavidin/scintillant-coated microplate (FlashPlate® PLUS; Perkin-Elmer Life Sciences). Biotinylated peptides would be captured in each well through their interaction with streptavidin, thereby bringing any incorporated [3H]methyl in proximity of scintillant. The amount of methylated peptide was quantified by tracing the radioactivity (cpm) as measured by the TopCount NXTTM Microplate Scintillation and Luminescence Counter (PerkinElmer Life Sciences). |
| Affinity data for this assay | |
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