利用双向电泳技术分离节瓜叶片总蛋白的方法研究
An optimization study on two-dimensional electrophoresis for chieh-qua leaf proteomic analysis
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摘要: 【目的】建立适合节瓜叶片蛋白质组学研究的双向电泳体系.【方法】以节瓜品种“A37毛节瓜”为材料，对蛋白质抽提方法、上样量、凝胶浓度及 IPG 胶条 pH 范围等关键因素进行探索与优化.【结果和结论】与TCA/丙酮沉淀法及酚提取法相比，改良酚提取法抽提蛋白质总量较高，纯度最高，SD-PAGE 电泳条带明显、清晰，双向电泳图谱蛋白质点最多、清晰均匀、纵横条纹少.利用17 cm pH 4~7范围 IPG胶条、120 μg蛋白质上样量、12%凝胶浓度，硝酸银染色获得蛋白质点数丰富、分辨率高、背景清晰且重复性好的双向电泳图谱，检测到节瓜叶片蛋白质点 1 936 个，相对分子质量主要分布于15 000~100 000之间.初步建立了一套适用于节瓜叶片蛋白质组学研究的双向电泳体系，为后续开展蛋白质组学研究及其相关工作奠定了基础.Abstract: 【Objective】To establish two-dimensional electrophoresis(2-DE) system suitable for chieh-qua leaf proteomics analysis. 【Method】The effects of various protein extraction methods of 2-DE were intercompared using the leaves of “A37maojiegua” as the materials, and the loading quantity of sample，SDS-PAGE gel concentration and the pH range of IPG strips were also investigated and optimized. 【Result and conclusion】The results showed that modified phenol extraction method was notably superior to TCA/acetone precipitation method and phenol extraction method in total protein quality and clarity of protein bands in SDS-PAGE electrophoresis, and the final 2-DE image map based on modified phenol extraction method had the maxium protein dots and clear background, slight streaking, more completely separated spots compared with the other methods. The further optimization of 2-DE system was carried out to obtain a 2-DE electrophoretogram abundant protein spots, high resolution, clear background and excellent repeatability with 17 cm IPG strips,120 μg protein loading quantity, SDS-PAGE with 12% gel concentration and IPG strips with pH 4-7, finally detecting proteins with modified silver nitrate staining. More than 1 936 protein spots were detected and their relative molecular mass ranged from 15 000 to 100 000. The optimization of the experimental conditions of 2-DE system suitable for chieh-qua proteomics research has been constructed, which provides a basis for further investigation of chieh-qua proteomics researches.
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Keywords:
- Chieh-qua /
- total protein of leaf /
- proteomics /
- two-dimensional electrophoresis
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