Protein isolation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE)

Plant total proteins were extracted from 20 mg of frozen ground leaf material homogenized in 100 µL of extraction buffer [25 mM BisTris/HCl (pH 7.0), 20% [v/v] glycerol, 0.25 g·L⁻¹ complete protease inhibitor (Roche)]. Membranes were solubilized by addition of 1% [w/v] n-dodecyl-β-d-maltoside (Sigma Aldrich) in the darkness on ice for 5 min. Finally, samples were denatured in sample buffer (62 mM Tris/HCl, pH 6.8, 10% [v/v] glycerol, 2% [v/v] sodium dodecyl sulfate, 0.0025% [w/v] bromphenol blue) for 3 min at 95°C. Equal volumes of crude extracts were loaded on the gel, 20 µL of crude extract corresponds to 100%. SDS–PAGE with 9% [T] separation gels was performed according to (Laemmli, 1970). After electrophoresis, proteins were visualized by Coomassie staining. Separation gels were incubated in staining solution (0.1% [w/v] Coomassie Blue G, 0.1% [w/v] Coomassie Blue R, 45% [v/v] methanol, 9% [v/v] acetic acid) for 10 min under continuous agitation at room temperature. Gels were destained by incubation in 30% [v/v] methanol, 5% [v/v] acetic acid solution.