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茭白线粒体蛋白双向电泳体系建立
作者: 杜传来1  罗海波2 *  彭 昕2  王韦华3  郁志芳3  
单位: (1 安徽科技学院 食品药品学院  安徽 凤阳 233100;2 浙江医药高等专科学校食品学院  浙江 宁波 315100;  
关键词: 茭白  线粒体  双向电泳  蛋白质组学  
分类号:Q81
出版年,卷(期):页码:2016 ,47 ( 3 ): 页码:332-336
摘要:

【目的】建立适合于茭白线粒体蛋白质组分析的双向电泳技术体系,为进一步开展茭白线粒体差异蛋白质组学研究提供参考。【方法】以茭白为试验材料,比较不同线粒体提取纯化方法、IPG胶条pH范围及蛋白上样量等条件对双向电泳图谱的影响。【结果】差速离心(3000~14000×g)联合Percoll不连续密度梯度离心(20%∶24%∶40%=2∶4∶2)对茭白线粒体的提取纯化效果优于仅采用差速离心。采用改良酚抽法提取茭白线粒体蛋白,17 cm、pH 3~10的IPG胶条,1.0 mg上样量,12% SDS-PAGE进行双向电泳,0.12%考马斯亮蓝G-250胶体考染法染色,茭白线粒体蛋白主要分布在pH 4~8范围内,pH 3~4和pH 8~10范围内蛋白点较少;进一步选用pH 4~7和pH 5~8的IPG胶条对茭白线粒体蛋白进行双向电泳,分离效果均明显提高,且pH 4~7范围内IPG胶条的双向电泳图谱清晰,蛋白点分辨率较高。分别采用0.8、1.0和1.2 mg 3个不同的上样量进行双向电泳,结果表明上样量为1.0 mg时,电泳图谱质量最好。【结论】通过差速离心联合Percoll不连续密度梯度离心提取纯化茭白线粒体,并控制好双向电泳体系的pH范围、上样量等因素,可获得蛋白质点清晰、分辨率高、重复性好的双向电泳图谱。

【Objective】The present experiment was conducted to establish two-dimensional electrophoresis(2-DE) system suitable for analysing mitochondrial proteomics of Zizania latifolia, in order to provide reference for further researching its differential proteomics of mitochondria. 【Method】Using Z. latifolia as material, the effect of mitochondrial extraction and purification methods, pH ranges of IPG strip, loading quantities, etc. on 2-DE map were compared and analyzed. 【Result】Based on comparison of Z. latifolia mitochondrial extraction and purification effect, the differential centrifugation(3000-14000×g) combined with Percoll discontinuous density gradient centrifugation(20%∶24%∶40%=2∶4∶2) was superior to single differential centrifugation method. The protein spots were mainly distributed in the range of pH 4-8 using the following procedure: extracting mitochondrial proteins with modified phenol extraction method, 17 cm pH 3-10 IPG strip, loading 1.0 mg protein sample, SDS-PAGE with 12% gel concentration, and finally staining with 0.12% coomassie brilliant blue(CBB) G-250, but the protein spots were less distributed in the ranges of pH 3-4 and pH8-10. The separation effect of 2-DE on mitochondrial proteins was improved significantly by using pH 4-7 and pH 5-8 IPG strips, and pH 4-7 was more suitable for separation of mitochondrial proteins because 2-DE map was more clear, and resolution of protein spots was higher. Furthermore, the 2-DE results of different protein loading quantities(0.8, 1.0, 1.2 mg) showed that, the quality of 2-DE map were the best at the loading quantity of 1.0 mg. 【Conclusion】The 2-DE map with clearer protein spots, higher resolution and good repeatability is obtained by extracting and purifying mitochondria with differential centrifugation combined with Percoll discontinuous density gradient centrifugation and controlling pH range and protein loading quantity.

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