小中大Phenol extraction of proteins
Phenol extraction of proteins is based on the protocol described before [8], but we carried out it in the presence of SDS (designated as phenol/SDS extraction). About 0.05–0.1 g of the dry powder of leaf tissue was resuspended in 0.8 mL phenol (Tris-buffered, pH 8.0; Sigma St. Louis, MO, USA) and 0.8 mL dense SDS buffer (30% sucrose, 2% SDS, 0.1 M Tris-HCl, pH 8.0, 5% 2-mercaptoethanol) in a 2.0 mL microtube. The mixture was vortexed thoroughly for 30 s and the phenol phase was separated by centrifugation at 10 000g for 3 min. The upper phenol phase was pipetted to fresh microtubes (0.2 mL for 1.5 mL tube, 0.4 mL for 2.0 mL tube). After phase separation, white SDS complex often appears at the interphase. Be careful not to disturb the interphase by pipetting. If the recovered phenol phase is not clear, pool phenol phase together and centrifuge again. At least 5 volumes of cold methanol plus 0.1 M ammonium acetate was added to the phenol phase and the mixture was stored at 20C for 30 min. Precipitated proteins were recovered at 10 000g for 5 min, and then washed with cold methanolic ammonium acetate twice and cold 80% acetone twice. The final pellet was dried and dissolved in a buffer of choice, such as Laemmli buffer [10] or 2-DE rehydration solution (8 M urea, 4% CHAPS, 2% IPG buffer, 20 mM dithiothreitol)
