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作 者:陈贤[1,2] 余初浪[2] 杨勇[2] 王栩鸣[2] 周洁[2] 张维林[1] 严成其[2] 陈剑平[2]
机构地区:[1]浙江师范大学化学与生命科学学院,浙江金华321004 [2]浙江省有害生物防控国家重点实验室培育基地,农业部植保生物技术重点实验室,浙江省植物病毒学重点实验室,浙江省农业科学院病毒与生物技术研究所,浙江杭州310021
出 处:《生物技术通讯》2012年第5期704-708,共5页Letters in Biotechnology
基 金:国家高技术研究发展计划(2008AAO2Z125);转基因新品种培育重大专项(2009ZX08001-006B;2011ZX08009-003-001);浙江省自然科学基金(Z307451)
摘 要:目的:建立适用于双向电泳分析的水稻悬浮细胞外分泌蛋白提取方法。方法:采用酚抽提结合甲醇醋酸铵沉淀法、三氯乙酸-丙酮沉淀法和硫酸铵沉淀等3种方法制备水稻悬浮细胞外分泌蛋白,并进行双向电泳分析;利用Western印迹对候选方法提取的外分泌蛋白进行纯度检测。另外,还利用质谱技术对从双向电泳胶上随机挑选的9个蛋白点进行测定,并用SignalP 3.0 Server对测定的蛋白点进行信号肽预测。结果:酚抽提结合甲醇醋酸铵沉淀法提取的外分泌蛋白得率最高,且双向电泳图谱清晰,并能检测到最多的蛋白点;Western印迹表明利用该法所提取的外分泌蛋白未被细胞内蛋白质污染。利用质谱技术鉴定了随机挑选的9个蛋白点,SignalP 3.0 Server分析表明其中6个蛋白含有信号肽。结论:酚抽提结合甲醇醋酸铵沉淀法是一种适用于双向电泳分析的水稻悬浮细胞外分泌蛋白提取方法。Objective: To establish an appropriate protein extraction for two-dimensional electrophoresis of secret- ed proteins from rice suspension cultured cells. Methods: Three methods to prepare samples for two-dimensional electrophoresis of secreted proteins from rice suspension cultured cells were tested: phenol extraction combined with methanol-ammonium acetate precipitation, trichloroacetic acid-acetone(TCA)-acetone precipitation and ammoni- um sulfate precipitation. The proteins isolated were tested by Western blot analysis to confirm if they were essen- tially free from contamination of non-secretory proteins. Ten random selected protein spots from 2-DE gels were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS). The signal peptide predict tool SignalP 3.0 was used to predict the presence of signal peptide cleavage sites in these proteins. Results: The phenol extraction combined with methanol-ammonium acetate precipitation resulted in the highest yield of secreted proteins and the most well-separated protein spots that could be detected on the 2-DE gel. Western blots confirmed that these secreted proteins were essentially free from contamination of non-secretory proteins. Six of the 9 protein spots selected by MALDI-TOF MS were predicted to contain signal peptide cleavage sites. Conclusion: It was concluded that phenol extraction combined with methanol-ammonium acetate precipita- tion was suitable for 2-DE analysis of secreted proteins from rice suspension cultured cells.
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