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蛋白质提取方法和电泳条件是蛋白质组学研究中双向电泳的关键。本文初步建立起一套适合圆果种黄麻功能叶总蛋白双向电泳(2-DE)分析的高效提取方法及电泳条件,在双向电泳技术中,比较了TCA丙酮、Tris-base/丙酮、Tris-HCl的蛋白提取方法,并优化了上样量和样品制备方法。结果表明,TCA丙酮法提取蛋白质产量高,达79.0mgg?1,比Tris-base/丙酮法和Tris-HCl的产量分别高出44.2%和114.1%。该法得到的2-DE图谱背景清晰,蛋白点数最多,约602个,较其他两种方法更适合用于黄麻功能叶总蛋白双向电泳分析;350μg为最佳上样量。这为黄麻蛋白质组学后续研究奠定了基础。
Protein extraction methods and electrophoresis conditions are the key to two-dimensional electrophoresis in proteomics research. In this paper, a set of high-efficiency extraction methods and electrophoresis conditions for 2-DE analysis of total protein of Juglans regia leaf were established. Two-dimensional electrophoresis was used to compare the effects of TCA acetone, Tris-base / acetone and Tris- HCl protein extraction methods, and optimize the sample volume and sample preparation methods. The results showed that the yield of protein extracted by TCA acetone method was 79.0mgg? 1, which was 44.2% and 114.1% higher than that of Tris-base / acetone and Tris-HCl, respectively. The 2-DE pattern obtained by the method has a clear background and a maximum of about 602 protein spots, which is more suitable for two-dimensional electrophoresis analysis of total leaf protein of jute function than the other two methods; and 350 μg is the optimal loading amount. This laid the foundation for follow-up study of jute proteomics.