| Assay Method Information | |
| | Microscale Thermophoresis (MST) |
| Description: | Kd represents dissociation constant, which measures the affinity of small molecule binding to the scaffold DDB1-BPB by Microscale Thermophoresis (MST) (see Table 1, column 9). MST experiments were performed on a Monolith NT 115 system (Nano Temper Technologies, Munich, Germany) using 100% LED and 60% IR-laser power (https://physiology.case.edu/media/eq manuals/eq_manual_nano-temper_mst_starting_guide.pdf, hereby incorporated by reference in its entirety for all purposes). The labeling of DDB1-BPB (10 M) was performed in labeling buffer with NHS-Red reactive dye (30 μM) (Nanotemper), which reacts efficiently with the primary amines of the proteins to form a stable dye protein conjugates. The labeling reaction was carried out at RT. A 16-point serial dilution (1:1) was prepared for compound at the final concentration ranged from 200 M to 3 nM in PBS containing 0.05% tween. The samples were filled into Premium capillaries and measurements were conducted at 25° C. An equation implemented by the software MO-S002 MO Affinity Analysis, provided by the manufacturer, was used for fitting normalized fluorescence values at different concentrations of ligands. |
| Affinity data for this assay | |
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