| Assay Method Information | |
| | Alpha-Synuclein Tissue Homogenate Binding Assay |
| Description: | For displacement α-synuclein binding assay, compounds and control were solvated in dimethyl sulfoxide (DMSO) and transferred using focused acoustic energy by an Echo 655 liquid handling instrument (Beckman Coulter, Indianapolis, IN) into designated wells of uniquely bar coded 96-well v-bottom low binding polypropylene microplates (Thermo Scientific, 249946). Compound dose response curves were prepared in a 10-point, 3-fold fashion within columns 2 -11 of the microplate from high to low compound concentrations. The final assay concentration of dose response curves when starting at 1 mM ranged from 1.2 µM to 0.061 nM (0.12 % DMSO final assay concentration, 270 nL/well). Controls included no inhibitor (DMSO only) dispensed into wells A1 – D1, A12 – D12 for minimum efficacy signal and Compound 1000 at a final assay concentration of 12 µM into wells E1 – H1, E12 – H12 for maximum efficacy signal. Liquid-handling steps for dispensing insoluble fractions of PD brain homogenates and 25666 radioligand were performed using a Bravo automated liquid handling platform equipped with a 96LT disposable tip head (Agilent Technologies, Santa Clara, CA). Insoluble fractions of PD brain homogenates were diluted to 50 µg/mL in the Assay Buffer, and 200 µL was dispensed to the assay plate for a final concentration of 10 µg/well. 25 µL of (9X) [3H]-1000 was dispensed to the assay plate for final assay concentration of 3.0 nM. Sealed assay plates were incubated at room temperature for 90 minutes with gentle agitation. The incubation was terminated by rapid filtration through UniFilter-96 GF/C microplates (pre-treated for 30 minutes with 0.2% Polyethylenimine at 4oC) by using a FilterMate Harvester (PerkinElmer). The microplates were subsequently washed four times with a total volume of 3.75 mL using ice-cold Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco 14190136 ) before drying 90 minutes at 47 oC with a vacuum oven (Fisher Scientific Isotemp 285A) or overnight at room temperature. The bottom of each UniFilter-96 GF/C microplate was adhesively sealed (PerkinElmer 6005199) prior to the addition of 50 μL MicroScint-20 liquid scintillation cocktail (PerkinElmer 6013621) to each well. A clear adhesive seal (TopSeal-A PLUS, PerkinElmer 6050185) was then applied to the top of each microplate and counted 1 minute/well on MicroBeta2 system (PerkinElmer, Model: 2450-0120). Data was analyzed using IDBS ActivityBase XE Runner (version 9.6.0.148) to determine Ki values shown in Data Table 1 (Kd value 0.90 nM, ligand concentration 3.0 nM). |
| Affinity data for this assay | |
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