一种适用于胡杨叶片非变性蛋白质提取的方法

A Novel Method for Nondenaturing Protein Extraction from Populus Euphratica Leaves

为探索适用于胡杨叶片非变性蛋白质的提取方法,以采自内蒙古额济纳旗天然胡杨林中胡杨成熟叶片为材料,采用咪唑法、Bis-Tris法、Tris-HCl法和新构建的Tris-SSAD法4种非变性蛋白质提取策略,分别用于提取胡杨叶片蛋白质样品;随后进行第一维非变性聚丙烯酰胺凝胶电泳(1^(st)-DE:Native-PAGE)。相比之下,只有Tris-SSAD法获得了清晰、条带数目较多的Native-PAGE图谱,且结合第二维变性凝胶电泳(2^(nd)-DE:SDS-PAGE)可成功分离出较多蛋白质复合体亚基或相互作用蛋白质分子,证明了Tris-SSAD法非变性温和提取特性。研究建立并优化了适用于胡杨叶片的非变性蛋白质提取方法,为后续进一步分析蛋白质与蛋白质相互作用及蛋白质复合体功能提供了实验基础。

Aims to establisii a nondenaturing protein extracted metiiod to study protein complexes and interacting proteins from leaves of Populus euphratia, four approaches, including Imidazole, Bis-Tris,Tris-HCI,and new method of Tris-SSAD （Tris based Sucrose,Sodium chloride, Ascorbate,and Digitonin solution ）,were applied in the nondenaturing extraction of leaf proteins. Based on the first-dimensional non-denaturing gel electrophoresis （1st- DE ： Native- PAGE）,it was found that the highest quality protein bands on CBB stained gel were only observed in the protein extracted by the method of Tris- SSAD. Further optimized processing was also performed to obtain more perfect results. Meanwhile, the nondenatuing feature of Tris-SSAD were also successfully proven by the tandem electrophoresis,composed of nondenaturing 1st-DE （ Native-PAGE） and denaturing 2nd-DE （SDS-PAGE ）,and a lot of protein complexes and/or interactive proteins were found in these electrophoresis gels. Finally,a suitable for the nondenaturing protein extraction from adverse-resistant plant leaves was achieved.