Table 2 Reagent set-up for real-time quaking-induced conversion

Homogenisation buffer. Filter-sterilised and stored in a 50 mL conical tube. Can be stored at 4 °C for up to 3 months.

SDS

0.05 g

0.1% (w/v)

1 × PBS pH 7.4

50 mL

Thioflavin-T solution. Prepared in conical tube and filter-sterilised. 10 mmol/L stock solution diluted to 1 mmol/L working solution. Can be stored in the dark at 4 °C or -20 °C for up to 3 months. Working solution should be discarded after use.

Thioflavin-T

0.036 g

10 mmol/L

ddH₂O

10 mL

–

5 × Phosphate buffered saline. Made up in 1L stock batch. Filter-sterilised and stored in a 50 mL conical tube. Can be stored at 4 °C for up to 3 months.

NaCl

KCl

Na₂HPO₄

KH₂PO₄

80 g

2 g

14.2

2.4 g

1.37 mol/L

26.8 mmol/L

0.1 mol/L

17.6 mmol/L

Base mix. For 5 reactions. Prepared in a conical tube and filter-sterilised. Can be stored at -20 °C for up to 3 months.

ddH₂O

217 µL

–

5 × PBS pH 6.9

100 µL

1.49 × 

5 M NaCl

17 µL

254 mmol/L

0.5 M EDTA

1 µL

1.49 mmol/L

Final reaction mix. Prepared in a conical tube. Prepare fresh for each experiment and gently mix using a P1000 pipette.

Base Mix

57 µL

1 × PBS, 170 mmol/L NaCl, 1 mmol/L EDTA

Thioflavin-T (1 mmol/L)

1 µL

10 µmol/L

Recombinant α-Syn monomer

27 µL

0.10 mg/mL

Note: total concentration of NaCl in final mix (PBS + 5 mol/L NaCl = 444.4 mmol/L)