| Assay Method Information | |
| | Fluorescence thermal melt assay |
| Description: | Thermal stabilities of CRBN-DDB1 in the presence or absence of phthalimide, thalidomide, lenalidomide and pomalidomide were done in the presence of Sypro Orange in a microplate format according to Pantoliano et al. Two ug of protein in 20 ul of assay buffer (25mM Tris HCl, pH 8.0, 150mM NaCl, 2 uM Sypro Orange) were subjected to stepwise increase of temperature from 20 to 70 °C and the fluorescence was read at every 1 °C on an ABIPrism 7900HT (Applied Biosystems, Carlsbad, CA, USA). Compounds were dissolved in DMSO (1% final in assay) and tested in quadruplicate at a concentration range between 30 nM to 1000 uM; controls contained 1% DMSO only. |
| Affinity data for this assay | |
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