| Assay Method Information | |
| | Time-Resolved Fluorescence Energy Transfer (Tr-Fret) Assay |
| Description: | Test compounds dissolved in DMSO were transferred by Echo 655T (Labcyte, San Jose, CA) to 384-well low-volume solid black plates. A mixture of biotin-KLHDC2, terbium-streptavidin (Thermo Fisher) and a Bodipy-labeled ligand probe (Lee-4893) was dispensed into the assay plates at a final volume of 20 μL using a Tempest (Formulatrix, Bedford, MA), followed by incubation at room temperature for 1 h prior to measurement of the TR-FRET signal using a PHERAStar plate reader (BMG Labtech, Durham, NC), which was equipped with modules for excitation at 337 nm and emission at 490 and 520 nm. The integration start was set to 100 μs, and the integration time was set to 200 μs. The number of flashes was fixed at 100. The 520/490 ratio was used as the TR-FRET signal in calculations. |
| Affinity data for this assay | |
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