Protein extraction and ammonium sulphate precipitation

Leaves from the transgenic plants (4-wk old) were frozen and crushed in liquid nitrogen. A protein extraction buffer (50 mM acetate buffer [pH 5.0], 500 mM NaCl, 0.5 mM EDTA, protease inhibitor cocktail for plant cell and tissue extracts [Sigma-Aldrich, St. Louis, MO, USA]) was added to each leaf sample. The leaf tissue suspension was centrifuged for 10 min at 15,000 × g at 4 °C. The supernatant was collected and neutralized with 1 M Tris-HCl (pH 8.0). After centrifugation, ammonium sulphate was added to the supernatant to 50% saturation on ice, and proteins were allowed to precipitate overnight at 4 °C. A sample was centrifuged for 10 min at 15,000 × g at 4 °C, and the precipitate was re-dissolved in PBS. Remaining insoluble aggregates were removed by centrifugation for 10 min at 21,500 × g at 4 °C. The supernatant was dialyzed against PBS with Mini Dialysis Kit 8 kDa cut-off (GE Healthcare, Buckinghamshire, UK). The resulting sample was used as the 50%-saturated ammonium sulphate precipitated fraction.