pET21a-alpha-synuclein

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	BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) were freshly co-transformed with pET21a-alpha-synuclein and pTSara-NatB and selected on ampicillin- (amp) and chloramphenicol- (cam) supplemented LB-agar plates. Cultures were grown in LB+amp+cam and induced at an OD600 of 0.5–0.6 with 0.2% (m/v) L-arabinose and, after 30 min, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, or with IPTG alone at an OD600 of 0.5–0.6).

Protocol tips

BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) were freshly co-transformed with pET21a-alpha-synuclein and pTSara-NatB and selected on ampicillin- (amp) and chloramphenicol- (cam) supplemented LB-agar plates. Cultures were grown in LB+amp+cam and induced at an OD600 of 0.5–0.6 with 0.2% (m/v) L-arabinose and, after 30 min, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, or with IPTG alone at an OD600 of 0.5–0.6).

Publication protocol

pET21a-alpha-synuclein was a gift from the Michael J. Fox Foundation MJFF (Addgene plasmid # 51486). BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) were freshly co-transformed with pET21a-alpha-synuclein and pTSara-NatB and selected on ampicillin- (amp) and chloramphenicol- (cam) supplemented LB-agar plates. Cultures were grown in LB+amp+cam and induced at an OD600 of 0.5–0.6 with 0.2% (m/v) L-arabinose and, after 30 min, with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, or with IPTG alone at an OD600 of 0.5–0.6). Growth was continued for 4 hrs. at 37°C under shaking. The cell pellet, after being harvested and kept frozen at -20°C overnight, was resuspended in 20 mM Tris buffer, 25 mM NaCl, pH 8.00, and lysed by boiling for 15 min. The supernatant of a 20-min, 20,000 x g spin of the lysate was then further processed. The sample was loaded on two 5-mL (tandem) HiTrap Q HP anion exchange columns (GE Healthcare, Pittsburgh PA), equilibrated with 20 mM Tris buffer, 25 mM NaCl, pH 8.00. αSyn was eluted from the columns with a 25–1000 mM NaCl gradient in 20 mM Tris buffer, 1 M NaCl, pH 8.00. For hydrophobic interaction chromatography αSyn peak fractions were pooled and injected on two 5-mL (tandem) HiTrap Phenyl HP hydrophobic interaction columns (GE Healthcare, Pittsburgh, PA), equilibrated with 50 mM phosphate buffer, 1 M (NH4)2SO4, pH 7.40. αSyn was eluted from the columns with a 1000–0 mM (NH4)2SO4 gradient in Milli-Q water. αSyn peak fractions were then pooled and further purified via size-exclusion chromatography on a HiPrep Sephacryl S-200 HR 26/60 column (GE Healthcare, Pittsburgh, PA) using 50 mM NH4Ac, pH 7.40 as running buffer. αSyn peak fractions were pooled, aliquoted, lyophilized and stored at -20°C.

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