适于茭白茎部蛋白质组分析的双向电泳技术体系的建立

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

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。

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.