三种核蛋白提取方法应用于双向电泳的比较

发布时间：2018-04-05 23:32

本文选题：核蛋白　切入点：蛋白质组学　出处：《浙江大学》2012年硕士论文

【摘要】：背景随着蛋白质组学的发展,人们对蛋白组学的研究已经深入到亚细胞水平,但是目前技术的分辨率对于全细胞蛋白质组学的分析无法满足人们的要求,于是亚细胞蛋白质组学的提出是蛋白质组学发展的必然结果。亚细胞蛋白质组学不仅可以降低蛋白的复杂性,而且可以富集低丰度蛋白,提高低丰度蛋白的检出率,有利于人们深入认识生命本质。细胞核蛋白质组学是亚细胞蛋白质组学的一部分,提取获得纯净的细胞核蛋白有利于细胞核蛋白质组学的研究。目的比较三种不同的真核细胞核蛋白提取方法制备的核蛋白应用于双向电泳的可行性及效率。方法 1.分别应用Dounce匀浆器研磨法、试剂盒提取法和涡旋破膜法提取HeLa细胞核蛋白； 2. Bradford法测定蛋白浓度； 3. Western blot检测标志性胞浆蛋白和核蛋白； 4.双向电泳后银染扫描图像,PDQuest8.0分析图像。结果涡旋法获得核蛋白的过程所需时间最短；试剂盒和涡旋法能够较快速的获得相对较纯的核蛋白；Dounce匀浆法获得的核蛋白量较少同时掺杂较多的胞浆蛋白。三种方法所获得的核蛋白双向电泳后其蛋白图谱基本相似。结论三种提取方法制备的核蛋白都可用于蛋白组学研究中的双向电泳分析,但试剂盒和涡旋法较Dounce法提取核蛋白纯度及效率高,且涡旋法更快速、成本更低。
[Abstract]:BackgroundWith the development of proteomics, the study of proteomics has gone deep into subcellular level, but the resolution analysis of whole cell proteomics can not meet the needs of people.So subcellular proteomics is the inevitable result of proteomics development.Subcellular proteomics can not only reduce the complexity of proteins, but also enrich low abundance proteins, improve the detection rate of low abundance proteins, and help people to understand the nature of life.Nuclear proteomics is a part of subcellular proteomics.PurposeThe feasibility and efficiency of three different methods for extracting eukaryotic nucleoprotein were compared.Method1.Dounce homogenizer grinding method, kit extraction method and vortex membrane breaking method were used to extract HeLa nuclear protein.2.Protein concentration was determined by Bradford.3.Western blot was used to detect the iconic cytoplasmic protein and nuclear protein.4.Silver staining scanning images were analyzed by PDQuest 8.0 after two-dimensional electrophoresis.ResultIt takes the shortest time to obtain the nucleoprotein by vortex method, and the relatively pure nuclear protein bounce homogenate method can obtain the relatively pure nuclear protein by the kit and vortex method, and the amount of nucleoprotein is less and the amount of cytosolic protein is more doped.The protein profiles obtained by the three methods were similar after two-dimensional electrophoresis.ConclusionAll the three methods can be used in the two-dimensional electrophoresis analysis of proteomics, but the purity and efficiency of the kit and vortex method are higher than that of Dounce method, and the vortex method is faster and lower cost.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

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