共查询到19条相似文献,搜索用时 171 毫秒
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发菜蛋白质组双向电泳技术的建立及优化 总被引:3,自引:0,他引:3
为建立适用于发菜(Nostoc flagelliforme)蛋白质组研究的双向电泳技术,对发菜蛋白质的提取、裂解、上样量、IEF及SDS-PAGE电泳等关键步骤进行了优化,结果显示:发菜蛋白质主要分布在pH 4~7范围内,采用改良TCA法可提高提取液中蛋白质的含量和双向电泳图谱的分辨率,裂解液含60 mmol/L DTT,24 cm IPG胶条上样量1.5 mg时不仅提高了蛋白质的溶解性,而且改善了双向电泳的分离效果,得到近800个蛋白点,且蛋白点清晰,图谱分辨率较好.采用优化后的双向电泳体系提高了发菜蛋白质双向电泳的分辨率和重复性,建立起一套适用于发菜蛋白质组分析的双向电泳方法. 相似文献
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莲(Nelumbo nucifera Gaertn.)不仅是重要的水生蔬菜作物之一,而且是进行基础研究的好材料。本文采用4种蛋白质提取方法(新型TCA/丙酮法、传统TCA/丙酮法、改良的Tris-HCl法、Tris-饱和酚法)并结合双向电泳技术,对莲子蛋白质提取方法进行筛选与优化。双向电泳实验结果显示,所得蛋白质图谱与莲种子蛋白质组成分布特点一致。通过PDQuest软件分析表明,新型TCA/丙酮法适用于莲子叶和胚芽组织的双向电泳蛋白质提取,而传统TCA/丙酮法则适用于莲胚轴组织双向电泳的蛋白质提取。研究结果为进一步利用质谱进行莲子蛋白质组研究奠定了基础。 相似文献
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种子蛋白质亚基结构快速测定双向电泳法 总被引:3,自引:1,他引:2
介绍了还原条件与非还原条件双向电泳法,并应用此方法研究了种子蛋白质亚基结构,实验结果表明,这种双向电泳法能快速地测定种子蛋白质的亚基结构。 相似文献
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建立并优化了沼泽型水牛睾丸曲精细管蛋白质分离的双向电泳体系,为后续水牛睾丸曲精细管蛋白质表达谱的鉴定和研究奠定基础。比较不同IPG胶条(线性与非线性)、胶条pH范围和蛋白质上样量这三个参数对双向电泳结果的影响。结果显示,采用上样量350μg的曲精细管总蛋白、24 cm且pH值4~7的线性IPG胶条能够更好地分离水牛睾丸曲精细管蛋白质,获得质量较好且分辨率较高的双向电泳图谱,得到大约486个蛋白点。双向凝胶电泳技术能够对水牛睾丸曲精细管蛋白质进行有效分离,并通过对双向电泳体系的优化可获得蛋白质点清晰且分辨率更高的双向电泳图谱。 相似文献
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为进行砂藓(Racomitrium canescens)蛋白质组学的双向电泳工作,首要前提是获得质量较好的砂藓总蛋白质,并建立一套完整高效的适用于砂藓的双向电泳体系。本研究以砂藓为研究对象,比较了Tris-酚抽提法、TCA-丙酮法和试剂盒法3种蛋白质提取方法获得砂藓总蛋白质的浓度和完整性,并对砂藓中蛋白质双向电泳的运行条件进行了优化和比较。结果表明:在3种砂藓总蛋白质提取方法中,Tris-酚抽提法获得的砂藓总蛋白质浓度最高,SDS-PAGE电泳结果中条带清晰且较完整,双向电泳后获得的蛋白质点最多,是3种方法中提取砂藓蛋白质浓度最高完整性最好的方法;基于Tris-酚抽提法的基础上进一步对砂藓总蛋白质双向电泳体系进行优化,结果显示:利用pH值范围为pH 4~7的IPG胶条,蛋白质上样量为1 000μg的条件下,获得的砂藓蛋白质双向电泳图谱蛋白质点最多,图谱中可分辨的蛋白质点共(872±15)个,且低丰度蛋白质点清晰。本研究筛选出的方法获得了蛋白质点分布均匀、背景清晰、分辨率高、质量较高的双向电泳图像,建立了砂藓蛋白质组学研究体系的较好方法。本研究结果为砂藓蛋白质组学研究提供了良好基础。 相似文献
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In proteomic research, two-dimensional electrophoresis (2-D) is an important tool for investigating differential patterns of qualitative and quantitative protein expression. The strength of the technique is due to its unrivalled power of being able to separate simultaneously thousands of proteins. The key to the comparison of 2-D protein profiles, however, lies in the use of a fast and robust image matching process which is essential to the subsequent quantification procedure. To satisfy the growing demand for a robust and fully automatic method of matching 2-D gel protein separation profiles, we describe in this paper a novel registration technique based on image intensity distribution rather than selected features. The method uses a multiresolution representation of the gel profiles and exploits the fact that coarse approximations to the optimal matching can be extracted efficiently from low-resolution images. This permits the removal of misalignments at different scales in a systematic manner and the strength of the new method has been confirmed by a double blind trial of 111 2-D gel pairs. The proposed method requires neither landmarks nor an a priori image alignment, and takes about five seconds for processing a typical gel pair on a standard personal computer. 相似文献
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Brookes PS Pinner A Ramachandran A Coward L Barnes S Kim H Darley-Usmar VM 《Proteomics》2002,2(8):969-977
The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed. 相似文献
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The resolving power of two-dimensional (2-D) gel electrophoresis has been tested using 17 allele products at five loci. Standard O'Farrell gels could separate 13 of these isozymes. The addition of a second pH gradient made it possible to separate all but one of the variant proteins. These results indicate that 2-D gel electrophoresis can resolve more than 90% of variants originally detected by one-dimensional (1-D) electrophoresis. The implications of these results for the low estimates of average heterozygosity obtained in surveys using 2-D gel electrophoresis are discussed. 相似文献
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Using 2-D electrophoresis, we analyzed proteins from transgenic rice overexpressing gibberellin acid (GA) catabolic enzyme, GA2-oxidase. These results indicate eight specific proteins differentially expressed in the transformed rice stems of T1 generation, but non in case of T2 generation. Proteins isolated from different stages of leaves of T1 generation showed no significant differences, except one-month-old leaf, where five differentially expressed proteins are visible.This work was supported in part by a research project of an Identification and Analysis of Proteins for Gene Discovery and Elucidation of Functions of Useful Genes in Rice Genome from Ministry of Agriculture, Forestry and Fisheries, and also supported by a part of grant from the Program of Basic Research Activities for Innovative Biosciences. Martin Hajduch was supported by European Commission’s specific research and technological development program “Confirming the International Role of Community Research” 1998-2002 (EU Fellowship to Japan). The authors are solely responsible for the content of this paper and it does not represent the opinion of the Community, and the Community is not responsible for any of use that might be made of date appearing herein. 相似文献
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植物蛋白质组学研究若干重要进展 总被引:1,自引:0,他引:1
植物蛋白质组学近年来正从定性向精确定量蛋白质组学的方向发展。国际上近两年发表的约160篇研究论文报道了利用不断改进的双向电泳结合生物质谱技术、多维蛋白质鉴定技术,以及包括双向荧光差异凝胶电泳、幅N体内代谢标记、同位素标记的亲和标签、同位素标记相对和绝对定量等在内的第2代蛋白质组学技术,对植物组织(器官)与细胞器、植物发育过程和植物响应环境胁迫的蛋白质组特征,以及植物蛋白质翻译后修饰和蛋白质相互作用等方面的研究成果。该文对上述报道进行总结,综述了2007年以来植物蛋白质组学若干重要问题研究的新进展。 相似文献