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1、蛋白质双向电泳总方案一、 植物蛋白的提取1 Chloroform/acetone method: This method was performed according to the protocol described by Xie et al. (2007), with modifications. The modifications included different tissue amount, reagent volume, and incubation time. These modifications also applied to the other three method
2、s described below.About 0.5 g of leaf or 1.5 g of root sample was homogenized in 4.0 mL of extraction buffer (0.1 M KCl, 0.5 M tris-base at pH 7.5, 0.05 M EDTA, and 2% 2-mercaptoethanol) for 10 min. The mixture was shaken on ice for 2 h and centrifuged at 12,000 g for 15 min at 4C. The supernatant w
3、as transferred to a new tube, and an equal volume of water saturated chloroform and isoamyl alcohol (24/1, v/v) was added. The tube was shaken at 4C for 30 min and then stored on ice for 10 min before centrifugation at 10,000 g for 10 min at 4C. The upper and bottom phases were removed. Then 4 mL of
4、 water was added to the interphase together with an equal volume of water-saturated chloroform and isoamyl alcohol (24/1, v/v); the mixture was homogenized, allowed to stand on ice for 10 min, and then centrifuged at 10,000 g for 10 min at 4C. The treatment was repeated twice. The interphase was was
5、hed with ice-cold acetone three times. The final pellets were vacuum-dried and dissolved in resolubilization solution (8 M urea, 2 M thiourea, 2% CHAPS, 1% dithiothreitol DTT, 1% pharmalyte).2 Phenol/ammonium acetate method: This method was developed following the protocol described by Hurkman and T
6、anaka (1986), with modification. About 0.5 g of leaf or 1.5 g of root sample was homogenized in 4.0 mL extraction buffer (50% phenol, 0.45 M sucrose, 25 mM EDTA, 1% v/v 2-mercaptoethanol, 250 mM tris-base, pH 8.8) for 10 min. Samples weretransferred to phenol-resistant screw-cap tubes and incubated
7、on a mixer for 30 min at 4C and then centrifuged at 5000 g for 10 min at 4C. The upper phenol-phase was removed and addedto five volumes of ice-cold 0.1 M ammonium acetate and 1% 2-mercaptoethanol in 100% methanol and then mixed before placing at 20C for 2 h. Precipitated protein was collectedby 10
8、min centrifugation at 12,000 g. Pellets were thoroughly washed twice with 20 mL of 0.1 M ammonium acetate in 100% methanol, followed by two washes with ice-cold 80% acetone,and a final wash with ice-cold 70% ethanol. The final pellets were vacuum-dried and dissolved in resolubilization solution.3 Tr
9、is-base/acetone method: This protocol was performed according to Rabilloud (1998), with modifi cation. About 0.5g of leaf or 1.5 g of root sample was homogenized in 4.0 mL of extraction buffer (40 mM tris-base, 5 M urea, 2 M thiourea,2% w/v CHAPS, 5% w/v polyvinylpyrollidone, and 2% 2-mercaptoethano
10、l). The homogenate was centrifuged for 15 min at 12,000 g. The protein in the supernatant was precipitated by adding four volumes of ice-cold acetone containing 0.07% (w/v) 2-mercaptoethanol, incubated at 20C for at least 2 h, and then centrifuged for 15 min at 12,000 g. The pellet was washed with i
11、ce-cold acetone containing 0.07%2-mercaptoethanol, incubated at 20C for 2 h, and centrifuged again at4C. The washing was repeated twice. The final pellets were vacuum-dried and dissolved in resolubilization solution.4 TCA/acetone method: This protocol was modified from the method described by Damerv
12、al et al. (1986). About 0.5 g of leaf or 1.5 g of root sample was homogenized for 10 min and incubated with 10 mL of precipitation solution (10% TCA and 0.07% 2-mercaptoethanol in acetone) for 2 h at-20C. The precipitated proteins were pelleted and washed with ice-cold acetone containing 0.07% 2-mer
13、captoethanol to remove pigments and lipids until the supernatant was colorless. The pellet was vacuum-dried, resuspended in resolubilization solution,and sonicated to extract proteins. Insoluble tissue was removed by centrifugation at 21,000 g for 20 min.5 Mg/NP-40: Two grams of plant tissues were p
14、laced in liquid nitrogen and then stored at 80. The plant tissue was transferred to a prechilled mortar, and ground with a pestle in liquid nitrogen to a fine powder. The powder was homogenized in 10 mL of ice-cold Mg/NP-40 extraction buffer containing0.5 M Tris-HCl, pH 8.3, 2% v/v NP-40, 20 mM MgCl
15、2, 2% v/v -mercaptoethanol, 1 mM phenylmethylsulfonylfluoride (PMSF) and 1% w/v polyvinylpolypyrrolidone(PVPP). After centrifugation at 12000g for 15 min at 4, proteins in the supernatant were precipitated by adding four volumes of cold acetone at 20 for 30 min for analysis of total protein by 2-DE.
16、注：除TCA/acetone外其它方法均为可溶性蛋白提取方法，在实验过程中发现用 protein extraction buffer提取后的蛋白液用预冷的TCA/acetone含0.07%-mercaptoethanol 沉淀效果比纯丙酮要好！因此，可以考虑其它蛋白提取方案与TCA/acetone结合。二、 蛋白的裂解1 裂解液的配方8 mol/L Urea,2 mol/L Thiourea,4 % CHAPS,1 % NP-40, 4 % TritonX-100,65 mmol/L DTT,0.5 mmol/L PMSF,0.5 % pharmalyte 3-10 8 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 20 mmol/L trisbase, 30 mmol/L DTE, 2% pharmalyte pH 3-10.8Murea,2M thiourea, 2%3-(3-cholamidopropyl)dimethylamonio-1-propanesulfonateCH
