小中大我的方法，有点过于繁琐了
Total protein was isolated from fully expanded mature leaves based upon a two-step procedure combining TCA/acetone precipitation and phenol extraction according to Wang et al. (2003), with some modification: (ⅰ) preparation of dry plant tissue power. Frozen leaf material (about 1 g) was pulverized to a fine powder in liquid nitrogen using a mortar and pestle, and then the powder was resuspended in a solution of 10% trichloroacetic acid (TCA) in acetone containing 0.07% 2-mercaptoethanol (2-ME). Proteins were allowed to precipitate for 1 h at −20°C, followed by centrifugation at 15000 g for 15 min at 4°C. The resultant pellet was sequentially rinsed with 10% TCA in acetone containing 0.07% 2-ME several times until the pellet was colorless, and then washed twice with cold acetone twice, and finally twice with cold 80% acetone. Each time the pellet was resuspended completely by vortexing and then centrifuged as above. The final pellet was dried under vacuum and used for phenol extraction of proteins or stored at -80°C for further use. (ⅱ)protein extraction. Phenol extraction of proteins is based on the protocol described by Hurkman and Tanaka (1986). The dried tissue powder was completely resuspended in 5 mL of extraction buffer (0.7 M sucrose, 0.5 M Tris-HCl, pH 7.5, 50 mM EDTA, 0.1 M KCl, 1% w/v polyvinylpyrrolidone (PVP), 2% v/v 2-mercaptoethanol, and 2 mM PMSF), by vortexing thoroughly for 30 min at 4°C. An equal volume of Tris-HCl saturated phenol pH 7.8 was added to the protein suspension. After an additional 30 min of shaking at 4°C, the phases were separated by centrifugation (16000 g for 30 min at 4°C). The phenol phase was collected and re-extracted with an equal volume of extraction buffer. Proteins were precipitated from the phenol phase by adding 5 volumes of 0.1 M ammonium acetate in methanol and incubated at least 2 h or overnight at -20°C. After centrifuging at 16 000g for 15 min (4°C), the collected protein pellets were washed three times with cold 0.1 M ammonium acetate in methanol, and then washed twice with cold 80% acetone. The final protein pellet was dried under vacuum at 4°C and resuspended in 500 μL of solubilization buffer (7 M urea, 2 M thiourea, 0.4% v/v Triton X-100, 4% w/v CHAPS, 1% dithiothreitol (DTT), 1% v/v IPG buffer pH 4–7) by incubating at room temperature for 1 h. Insoluble matter was removed by centrifugation for 20 min at 16,000g. Protein concentration was determined using Bradford assay (1976).
