本文選題：核蛋白　切入點(diǎn)：蛋白質(zhì)組學(xué)　出處：《浙江大學(xué)》2012年碩士論文

【摘要】：背景 隨著蛋白質(zhì)組學(xué)的發(fā)展,人們對(duì)蛋白組學(xué)的研究已經(jīng)深入到亞細(xì)胞水平,但是目前技術(shù)的分辨率對(duì)于全細(xì)胞蛋白質(zhì)組學(xué)的分析無(wú)法滿足人們的要求,于是亞細(xì)胞蛋白質(zhì)組學(xué)的提出是蛋白質(zhì)組學(xué)發(fā)展的必然結(jié)果。亞細(xì)胞蛋白質(zhì)組學(xué)不僅可以降低蛋白的復(fù)雜性,而且可以富集低豐度蛋白,提高低豐度蛋白的檢出率,有利于人們深入認(rèn)識(shí)生命本質(zhì)。細(xì)胞核蛋白質(zhì)組學(xué)是亞細(xì)胞蛋白質(zhì)組學(xué)的一部分,提取獲得純凈的細(xì)胞核蛋白有利于細(xì)胞核蛋白質(zhì)組學(xué)的研究。 目的 比較三種不同的真核細(xì)胞核蛋白提取方法制備的核蛋白應(yīng)用于雙向電泳的可行性及效率。 方法 1.分別應(yīng)用Dounce勻漿器研磨法、試劑盒提取法和渦旋破膜法提取HeLa細(xì)胞核蛋白； 2. Bradford法測(cè)定蛋白濃度； 3. Western blot檢測(cè)標(biāo)志性胞漿蛋白和核蛋白； 4.雙向電泳后銀染掃描圖像,PDQuest8.0分析圖像。 結(jié)果 渦旋法獲得核蛋白的過(guò)程所需時(shí)間最短；試劑盒和渦旋法能夠較快速的獲得相對(duì)較純的核蛋白；Dounce勻漿法獲得的核蛋白量較少同時(shí)摻雜較多的胞漿蛋白。三種方法所獲得的核蛋白雙向電泳后其蛋白圖譜基本相似。 結(jié)論 三種提取方法制備的核蛋白都可用于蛋白組學(xué)研究中的雙向電泳分析,但試劑盒和渦旋法較Dounce法提取核蛋白純度及效率高,且渦旋法更快速、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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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