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4/27/2019· In brief, cell lysis and protein extraction were carried out simultaneously by phenol extraction in a ball mill. Extracted proteins were dissolved in a 2-mL aqueous solution containing 7 M urea, 2 M thiourea, and 0.01 g mL −1 1,4-dithiothreitol. Amido black assay was used to quantify protein concentration [56, 57].

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After centrifugation (10 min, RT, 10,000 g), the upper phenol phase was collected into a new 15 mL reaction tube and washed with the same volume of 1 M sucrose solution for 10 min at RT and 120 rpm on a shaker/ball mill. Finally, the sample was centrifuged again (10 min, RT, 10,000 g), and proteins were precipitated by ammonium acetate in ...

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8/16/2019· Finally, the protein pellet was dried at 60°C for 15 min and dissolved in 1 mL urea buffer (7 M urea, 2 M thiourea, 1% DTT). After 10 min shaking in a ball mill at (RT, 1,800 rpm), non-dissolved particles were removed by centrifugation (10 min, 4°C, 10,000 g). …

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