Hiload 26 60 superdex 75 preparation grade

Recombinant HαSyn was purified in E. coli using plasmid pT7-7 encoding for the protein as previously described [7 (link)].
The expression was induced with 1 mM IPTG at 37 °C for 4 h. The cell lysate was centrifuged at 22,000× g (Beckman Coulter, Brea, CA, USA) for 30 min and the supernatant was then heated for 20 min at 70 °C. After centrifugation at 22,000× g, two steps of precipitation and centrifugation were employed and in particular 10 mg·mL⁻¹ streptomycin sulfate was added to the supernatant for DNA precipitation and, subsequently, 360 mg·mL⁻¹ ammonium sulfate was added to the supernatant to precipitate the recombinant HαSyn. The obtained pellet was resuspended in 25 mM Tris-HCl, pH 7.7 and, after dialysis against the same buffer, loaded onto an anion exchange column (26/10 Q sepharose high performance, GE Healthcare, Little Chalfont, UK) to be eluted with a 0–1 M NaCl step gradient. Further purification was obtained with size exclusion chromatography (Hiload 26/60 Superdex 75 preparation grade, GE Healthcare). The purity of the sample was analyzed by SDS-PAGE and the protein concentration was determined from the absorbance at 275 nm using an extinction coefficient of 5600 M⁻¹·cm⁻¹.

Recombinant human αSyn (H-αSyn) was purified in E. coli using plasmid pT7-7 encoding for the protein as previously described [30 (link)]. The expression was induced with 1 mM IPTG at 37 °C for 4 h. The cell lysate was centrifuged at 22,000 g (Beckman Coulter, Brea, USA) for 30 min, and the supernatant was then heated for 20 min at 70 °C. After centrifugation at 22,000 g, two steps of precipitation and centrifugation were employed and, in particular, 10 mg·mL⁻¹ streptomycin sulfate was added to the supernatant for DNA precipitation. Subsequently, 360 mg·mL⁻¹ ammonium sulfate was added to the supernatant to precipitate the recombinant H-αSyn. The obtained pellet was resuspended in 25 mM Tris–HCl, pH 7.7 and, after dialysis against the same buffer, loaded onto an anion exchange column (26/10 Q sepharose high performance, GE Healthcare, Little Chalfont, UK) to be eluted with a 0–1 M NaCl step gradient. Further purification was achieved by applying size exclusion chromatography (Hiload 26/60 Superdex 75 preparation grade, GE Healthcare). The purity of the sample was analyzed by SDS-PAGE, and the protein concentration was determined from the absorbance at 275 nm using an extinction coefficient of 5600 M⁻¹·cm⁻¹.