摘要
为给野生大豆蛋白质组学分析提供技术支撑,以耐盐碱野生大豆叶片为材料,对Tris-饱和酚法、Tris-HCl法和TCA-丙酮法3种蛋白质提取方法进行了比较,并对蛋白上样量、等电聚焦参数及凝胶浓度进行了优化。结果表明:使用Tris-饱和酚法提取蛋白质,上样量为2 000μg,等电聚焦90 000 V·h,凝胶浓度为10%时,可获得背景清晰、蛋白点数较多、重复性较好的蛋白双向电泳图谱。
To facilitate the proteomic analysis of wild soybean leaves, in this study, we established a suitable protocol for leaf protein extraction and two-dimensional electrophoresis. We extracted the total proteins of Glycine soja leaves through Tris-phe- nol method, Tris-HC1 method and TCA-acetone method, compared the protein quantity and yield, and found that proteins ex- tracted by Tris-phenol method displayed the highest concentration and best quality. We also compared the 2-DE images ob- tained by using different protein loading amounts, isoelectric focusing （IEF）parameters, and gel concentrations. Our results i- dentified a suitable protocol for 2-DE analysis, using the Tris-phenol method for protein extraction, 2 000 μg protein loading amount, 90 000 V · h IEF condition, 10% gel concentration. Under this condition, we obtained good 2-DE images with much clearer background, more protein spots, and better reproducibility. Taken together, in this study, we establisheda suitable system for 2-DE analysis of wild soybean leaf proteins, which will lay a foundation for the following Glycine soja leaf proteomic research.
出处
《大豆科学》
CAS
CSCD
北大核心
2015年第5期874-880,共7页
Soybean Science
基金
国家自然科学基金(31171578)
国家转基因重大专项(2011ZX08004)
黑龙江省高校科技创新团队建设计划(2011TD055)
关键词
野生大豆
叶片
蛋白质提取
双向电泳
蛋白组学
Glycine soja
Leaves
Protein extraction
Two-dimensional electrophoresis
Proteomics
