| Assay Method Information | |
| | Determination of Binding Affinity to EBP-7-Dehydrocholesterol Reductase |
| Description: | Compounds were prepared in a 96-well U bottom plate (Corning Cat #7007) using an Echo550 machine and 10 mM compound DMSO stock solution, followed by an 8-dose 5-fold serial dilutions protocol with final testing compound concentration ranging from 0.06 to 5000 nM, with DMSO back fill to 100 nL/well and n=2. DMSO and Ifenprodil (Sigma, Cat #12892) 5 μM wells were added in each plate as 0 and 100% inhibition reference controls with n=8 for each condition. The UniFilter-96 GF/B plates (PerkinElmer Cat #6005177) were pre-treated by adding 50 μl/well of 0.3% (v/v) Polyethylenimine (PEI) (branched, Sigma Cat #408727) to UniFilter-96 GF/B plates. The plates were sealed and incubated at 4° C. for 3 hrs. Then, the plates were washed with ice-cold assay buffer 3 times. The radioligand binding assay was prepared by adding assay buffer diluted hEBP-DHCR7 membrane at 66.7 g/ml×150 μl/well into the 96-well compound plate to reach 10 μg membrane per well. Then, the assay buffer diluted [3H]—(S)-6-(2-Methyl-3-(6-(trifluoromethyl)pyridin-3-yl)propyl)-2-thia-6-azaspiro[3.4]octane 2,2-dioxide (Moravek, Cat #MT-1003106) was added at 25 nM×50 μl/well. Following this, the plate was centrifuged at 1000 rpm for 30 secs. The plate was then sealed and agitated at 600 rpm at 22° C. for 5 min, and then incubated at 22° C. for 3 hrs. The incubation was stopped by transferring the binding solution to the pre-treated UniFilter-96 GF/B plate, vacuum filtrated, and then washed four times with ice-cold assay buffer. Following this, the plates were dried at 37° C. for 45 min. The plates were then sealed at the bottom. 40 l/well of scintillation cocktail was added to the plates. |
| Affinity data for this assay | |
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