| Assay Method Information | |
| | Q46 Radioligand Binding Assay |
| Description: | For radioligand binding assays (RBA) MBP-HTT(1-89)Q46-His(6 ) ( Exonl-Q46 ) protein was generated based on a previous publication (Scherzinger et al. Cell, Vol. 90, 549-558, Aug. 8, 1997). For experiments 30 uM MBP-Exonl-Q46 was incubated with 150 ug/mL thrombin in assay buffer (150 mM NaCl, 50 mM Tris pH 8.0) and 2 mM CaCl2 for 16 hours at 37 C. Aggregated Exonl-Q46 was pelleted by centrifugation for 5 minutes at 13,000 rpm in a bench top centrifuge and re-dissolved in the same volume of assay buffer. Test compounds were prepared by titration in DMSO at 11 concentrations from 63 uM to 2 nM. For the RBA, Q46 protein aggregates and test compounds were pre-incubated in assay buffer for 20 minutes at room temperature, in 100 uL/well in a 96-well plate (pp, round bottom). Then, ligand was added in 50 uL/well and incubated for 60 minutes at 37 C. Final assay concentrations were 1 uM to 30 uM test compound, 1 uM Exonl-Q46 protein (equivalent monomer concentration) and 0.3 nM ligand [3H3-methyl]-5-((5-methoxypyridin-2-yl)methoxy)-2-(pyrazin-2-yl)benzo[d]oxazole. Samples were transferred onto GF/B filter plates and washed 2 with 200 uL PBS using a Filtermate Harvester. After drying filter plates for 1 hour at 55 C., the back of the plates were sealed with foil and 30 uL/well scintillation fluid (Packard MicroScint 40) added, incubated for 15 minutes in the dark and counted in a MicroBeta reader. For analysis, replicate data from independent assay plates were normalized towards 0% and 100% inhibition using control wells of vehicle (0% inhibition) and 1 uM unlabelled [3H3-methyl]-5-((5-methoxypyridin-2-yl)methoxy)-2-(pyrazin-2-yl)benzo[d]oxazole (100% inhibition). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |