›› 2010, Vol. 22 ›› Issue (3): 0-286.

• 论文 •    

适于茭白茎部蛋白质组分析的双向电泳技术体系的建立

刘倩1,阮松林2,俞晓平1,马华升2,叶子弘1,*   

  1. 1中国计量学院 生命科学学院,浙江省生物计量及检验检疫技术重点实验室,浙江 杭州 310018;2杭州市农业科学研究院 生物技术研究所,浙江 杭州 310024
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-05-25 发布日期:2010-05-25

A two-dimensional electrophoresis protocol suitable for proteomic study of stem in Zizania latifolia Turcz.

LIU Qian;RUAN Song-lin;YU Xiao-ping;MA Hua-sheng;YE Zi-hong;*   

  1. 1College of Life Sciences, Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, China Jiliang University, Hangzhou 310018, China; 2Institute of Biotechnology, Hangzhou Academy of Agriculture Science, Hangzhou 310024, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-05-25 Published:2010-05-25

摘要: 对茭白茎部总蛋白的提取方法,以及2-DE的条件进行了摸索比较,以期得到适合于茭白茎部组织双向聚丙烯酰胺凝胶电泳的方法。结果表明:应用三氯乙酸(TCA)—丙酮沉淀法在研磨时加入聚乙烯吡咯烷酮(PVP),适当增加离心、沉淀的次数和时间,并且提高裂解浓度,可以得到浓度较高,质量较好的蛋白质样品;在蛋白含量测定中减少误差,对2-DE方法中上样量的多少、等电聚焦的程序进行优化,比较并选择染色方法,最终得到蛋白质点数目多,背景清晰,条纹较少,拖尾现象不明显的电泳图谱。

关键词: 茭白, 双向电泳, 蛋白质组

Abstract: In search of a method that was adapt to two-dimensional electrophoresis of Zizania latifolia stem, we compared different methods of protein extraction. Optimized conditions for 2-DE were carried out. The results showed that the protein sample of higher concentration and better quality could be acquired if we made proper changes in extraction of TCA-acetone method. In the process, 10% PVP was added in grinding, the centrifugation time was extended to 20 min and the number of precipitation time increased to 4 or 5, meanwhile, lysate was also condensed. We also decreased errors in protein assaying. During the optimization of 2-DE, changes of sample quantity, process of isoelectro focusing(IEF)and the method of staining became important factors. Compared with coomassie brilliant blue G-250 staining, silver ammonia staining detected much more spots. All of the efforts got a better result with more spots, less streak, clearer background and more unconspicuous tailing.

Key words: Zizania latifolia Turcz., two-dimensional electrophoresis (2-DE), proteome