Alpha-synuclein protein assay protocol

Protocol for assaying active alpha-synuclein monomer and aggregate binding to thioflavin T.

1. Prepare 1 mM stock solution of Thioflavin T (ThT) in dH₂O (prepare fresh and filter through a 0.2 μm syringe filter).

2. Dilute the ThT in PBS (pH 7.4) so that the final ThT concentration in each well is 25 μM.

3. Thaw the alpha-synuclein (monomer and aggregate) aliquots on ice just before use.

4. Either add 10 µM aggregate or 100 μM monomer (or both) to the appropriate wells.

5. Seal and place the plate in a shaking incubator (600 rpm) at 37°C.

6. Measure ThT fluorescence using a fluorescence microplate reader (excitation 450 nm, emission 485 nm) at 37°C from 1 hour up to 72 hours.

ThT emission curves show increased fluorescence (correlated to alpha-synuclein protein aggregation) over time when 10 µM of active alpha-synuclein aggregate (ab218819) is combined with 100 µM of active alpha-synuclein monomer (ab218818) (light blue), as compared to when 100 µM of active alpha-synuclein monomer is combined with 10 µM of control alpha-synuclein aggregate (purple line), or 100 µM of control alpha-synuclein monomer (ab218816) is combined with 10 µM of control alpha-synuclein aggregate (ab218817) (dark blue). ThT ex = 450 nm, em = 485 nm.

Figure 1. Graph showing ThT fluorescence following interactions with alpha-synuclein monomers, aggregates and controls.

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