Thioflavin T alpha-synuclein aggregation assay

Fibrillation assay for aS was set up in a 96-well black plate (Perkin Elmer) with flat black bottom. For the control samples, lyophilized protein was dissolved in PBS (10 mM sodium phosphate, 1.8 mM potassium phosphate pH 7.4 and 137 mM NaCl, 2.7 mM KCl) and passed through a 100 kDa MWCO centrifugal filter unit (Amicon^® Ultra, Millipore) to retain unsolved aggregates that can act as seeds. Protein concentration was determined using a calculated extinction coefficient (Abs 0.1%) at 280 nm in water of 0.412 ml/(mg*cm); for the modified protein the concentration was referred to the alpha-synuclein before DOPAL reaction and filtration was avoided because reacted material was retained by the filter. For each protein were prepared 3 wells: each well contains 200 μl of sample, constituted of 1 mg/ml (about 70 μM) protein, 20 μM ThT, 0.05% sodium azide. The plate was sealed with MicroAmp^® Optical Adhesive Film (Applied Biosystems^® - Life technologies) to avoid evaporation and incubated into a thermostated orbital shaker (Talboys Professional Model 1000MP Microplate Shaker by Troemner) at 37 °C at 1000 rpm. Aggregation progression was monitored measuring ThT emission with a VICTOR™ X3 Multilabel Plate Reader (PerkinElmer) at indicated intervals of time. ThT fluorescence was measured with excitation at 450 nm and emission at 535 nm, with a measurement time of 0.1 s, a CW-lamp energy of 5000 and a small emission aperture.