Thioflavin T assay protocol for alpha-synuclein proteins

Last edited Wed 01 Feb 2023

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.

Products used in this protocol

Method

Steps

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

• Prepare fresh in dH₂O
• Filter through a 0.2 μm syringe filter

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

Thaw the alpha-synuclein aliquots to room temperature just before use. This includes both monomer and aggregate.

Add either 10 µM aggregate or 100 μM monomer to the appropriate wells.

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

Measure ThT fluorescence using a fluorescence microplate reader.

• The reader should be set to excitation 450nm and emission 485nm.
• Read at 37°C from 1 hour up to 72 hours.