| Assay Method Information | |
| | TR-FRET Assay |
| Description: | The TR-FRET assay was carried out in black 384-well microtiter plates at a final volume of 20 μM per well. To screen library compounds, the assay cocktail was prepared as a mixture of 50 nM biotin-DCN1, 20 nM Ac-UBE2M12-AlexaFluor488, 2.5 nM Tb streptavidin (ThermoFisher) in assay buffer (25 mM HEPES, 100 mM NaCl, 0.1% Triton X-100, 0.5 mM DTT, pH 7.5). The assay cocktail was then incubated for 1 h at room temperature and distributed using a WellMate instrument (Matrix). Compounds to be screened were added to assay plates from DMSO stock solutions by pin transfer using 50SS pins (V&P Scientific). The assay mixture was incubated for 1 h at room temperature prior to measuring the TR-FRET signal with a PHERAstar or Clariostar plate reader (BMG Labtech)) equipped with excitation modules at 337 nm and emissions at 490 and 520 nm. We set the integration start to 100 s and the integration time to 200 s. The number of flashes was set to 100. The ratio of 520:490 was used as TR-FRET signal in calculations. Assay end points were normalized from 0% (DMSO only) to 100% inhibition (unlabeled competitor peptide) for hit selection and curve fitting. All compounds were tested in triplicate or more. |
| Affinity data for this assay | |
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