| Assay Method Information | |
| | Alpha-Synuclein Competitive Binding Assay |
| Description: | (A) Expression and purification of recombinant wild-type human α-synuclein: 0.5 mM IPTG was used to induce production of wild type human α-synuclein by bacteria transformed with a full-length α-synuclein expression plasmid. After shaking at 16° C. for 20 hours, cell pellet was resuspended in lysis buffer (10 mM Tris, 1 mM EGTA, 0.75 mM NaCl, 1 mM PMSF, pH 7.5) and lysed by sonication followed by centrifugation (6000 g, 30 min, 4° C.). The supernatant was then boiled at 95° C. for 15 min with manual agitation by every 5 minutes, followed by centrifugation (6000 g, 30 min, 4° C.). Supernatant was dialyzed with (10 mM Tris, 1 mM EGTA, 50 mM NaCl, pH 7.5) buffer and concentrated with concentrator. The collected sample was loaded onto a Superdex200 column, the flow-through fraction was then loaded onto a Q-HP column. Collected fractions with α-synuclein eluates were pooled, concentrated and stored at −80° C.(B) Preparation of aggregated α-synuclein: 5 mg/mL of α-synuclein in PBS buffer (pH 7.4) was incubated in tube with at 37° C. with shaking (700 rpm) for 5-10 days.(C) In vitro fluorometric α-synuclein binding assays: 2 μM α-synuclein was incubated with serially diluted compound (three-fold serial dilutions, from 10 to 0.001 μM) in a 96-well plate at 37° C. for 1 hour. Fluorescence intensity was read by microplate spectrometer. Compound Kd values were calculated using the following equation: Y=Bmax*X/(Kd+X), where X is the concentration of compound; Y is the fluorescence signal of (compound+α-synuclein)−(compound+DMSO); and Bmax is the maximum signal. |
| Affinity data for this assay | |
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