α-syn purification

Hello, 

Here is the protocol. If you have specific questions, feel free to email me. 

a-syn purification
The a-syn construct was transformed into Escherichia coli expression cell line BL21 (DE3) gold (Agi- lent Technologies). For expression, 10 ml LB + Amp (100 mg/mL) was inoculated from transformed colonies and grown overnight. 30 ml of starting culture was added to a 2 L flask containing 1 L LB + Amp (100 mg/mL) and grown for 3 hr at 37 ̊C to OD600 = 0.6. IPTG was then added to 0.5 mM to induce protein expression, which continued for an additional 3 hr. The bacterial pellet was collected by centrifugation at 4000 rpm for 10 mins. Cell pellet was resuspended in 15 mL/L pellet lysis buffer 

(100 mM Tris- HCl pH 8.0, 1 mM EDTA pH 8.0) and lysed by sonication. Crude cell lysate was clari- fied by centrifugation at 15000 g for 30 min at 4 ̊C. 10 mg/ml Streptomycin was added to the super- natant and stirred on ice for 30 mins followed by centrifugation at 15000 rpm for 30 mins. Protein was then purified by ammonium sulfate precipitation by adding 0.22 g/ml ammonium sulfate and stirred on ice for 30 mins followed by centrifugation at 15000 rpm for 30 mins. The supernatant was discarded and the pellet re-suspended in 12 mL/L pellet of 20 mM Tris pH 8.0. The solution was then dialyzed against 4 L 20 mM tris pH 8.0 overnight to remove residual ammonium sulfate. Next day, the protein was purified by HiPrep Q HP 16/10 column (GE Healthcare) using buffer A (20 mM Tris pH 8.0) and buffer B (20 mM Tris pH 8.0; 0.5M NaCl) using a gradient from 0–100% buffer B over 100 mL. Fractions containing protein were collected and pooled and injected on a preparative size exclusion silica G3000 column (Tosoh Bioscience). The column buffer comprised 0.1 M sodium sulfate, 25 mM sodium phosphate, and 1 mM sodium azide, pH 6.5. The purified protein was dia- lyzed in 0.1 M sodium sulfate and 25 mM sodium phosphate twice for 4 hr and 12 hr to remove sodium azide. Protein fractions were collected and concentration measured by Pierce BCA protein assay (Thermo #23225).

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