银杏种胚蛋白提取及双向电泳体系的优化

Embryo Protein Extraction from Ginkgo biloba and Optimization of Two-Dimensional Electrophoresis

[目的]探索一套适用于银杏种胚蛋白的双向电泳体系,为研究银杏种胚在不同发育阶段的表达差异蛋白提供基础。[方法]分别从蛋白的不同提取方法(直接提取法、酚提取法、Tris-HCl法、Trizol提取法和TCA-丙酮-酚法)、裂解液中的尿素浓度(6 mol·L^(-1)、7 mol·L^(-1)、8 mol·L^(-1)、9 mol·L^(-1))蛋白上样量(1 500μg、1 650μg、1 800μg)和分离胶浓度(10%、12.5%、15%)四个方面对银杏种胚的蛋白图谱进行分析,筛选出适宜双向电泳的条件。[结果]采用TCA-丙酮-酚法提取银杏种胚蛋白,在裂解液中尿素浓度为8 mol·L^(-1)、上样量为1 650μg、分离胶浓度为12.5%的方法,可以获得分辨率较高、背景更加清晰、重复性更好的银杏种胚蛋白质双向电泳图谱。[结论]建立了适合银杏种胚蛋白双向电泳的体系:TCA-丙酮-酚法制备样品,选取尿素浓度为8 mol·L^(-1)的裂解液提取样品粉末全蛋白,蛋白上样量控制为1 650μg,第二向分离胶浓度定为12.5%。

Objective]A two-dimensional （2-DE ）electrophoresis system was explored for proteomic analysis of Ginkgo biloba embryo,aiming at laying a foundation for studying the differences in protein expression at different de-velopmental stages.[Method]The 2-DE gels were involved with different protein extraction methods （direct extrac-tion method,phenol extraction method,Tris-HCl method,Trizol extraction and TCA-acetone-phenol method）,dif-ferent concentrations of urea in lysis buffer （6 mol·L^-1 ,7 mol·L^-1 ,8 mol·L^-1 ,and 9 mol·L^-1 ）,different protein loading dosages （1 500 μg,1 650 μg,and 1 800 μg）and different separation gel concentrations （10%, 12.5%,and 15%）,to select the suitable condition.[Result]The optimized system includes the following steps：extracting total protein from G.biloba embryo by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L^-1 urea,1 650 μg of protein loading dosage and running SDS-PAGE with 12.5%gel concentra-tion.Reproducible profiles with high resolution and clean background were obtained by this optimized two dimen-sional electrophoresis of embryonic total protein.[Conclusion]The optimized 2-DE electrophoresis system suitable for the separation of embryonic total protein from G.biloba was established,which is：extracting total protein by TCA-acetone-phenol method,dissolving proteins with lysis buffer containing 8 mol·L^-1 urea,1 650 μg of protein loading dosage and running SDS-PAGE with 12.5% gel concentration.