权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of high-performance techniques for the separation and analysis of minor protein components in human plasma

开发用于分离和分析人血浆中微量蛋白质成分的高性能技术

基本信息

批准号：
11640610
负责人：
MANABE Takashi
金额：
$ 0.51万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

In the course to analyze total proteins in human plasma and lymphocytes, we have used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) since it has the best resolution for the analysis of proteins. However, most of the workers on total protein analysis are using 2-D PAGE in denaturing conditions, to obtain structural information of the polypeptides separated on the 2-D gels. However, ultimate aim of protein analysis must be the re-construction of protein functions in living cells or organs. For this purpose, we have been using 2-D PAGE in the absence of denaturants. In this research project, we proposed a combination of four 2-D PAGE techniques (Type-I, Type-II, Type-III and Type-IV) to correlate structural information of polypeptides (obtained by Type-IV 2-D PAGE) with information on protein functions (obtained by Type-I 2-D PAGE).Further, we developed new electrophoretic techniques of protein separation and recovery which overcome the shortage of conventional 2-D PAGE te … More chniques. In conventional 2-D PAGE, after electrophoresis proteins are fixed on the slab gel, stained with a dye or silver nitrate, quantitated by an image analysis software installed in a microcomputer, and extracted from the gel piece for further structural analysis. Most of these procedures require manual operation and their automation is almost impossible. During 1996-1998, we have developed techniques of capillary electrophoresis for the separation of proteins. Capillary electrophoresis is characterized by on-column detection, on-column quantitation, and full-automation of the separation process. We established the conditions of capillary isoelectric focusing and capillary SDS electrophoresis and applied them for the separation of human plasma proteins. Then we extended these conditions of capillary electrophoresis aiming to establish high-performance techniques for protein separation and recovery. We have constructed several apparatus and devises for this purpose and we are evaluating their performance in the separation of minor protein components in human plasma. Less

在分析人血浆和淋巴细胞总蛋白的过程中，我们使用了二维聚丙烯酰胺凝胶电泳法(2D PAGE)，因为它对蛋白质的分析具有最好的分辨率。然而，大多数从事总蛋白分析的工作人员都是在变性条件下使用二维PAGE，以获得在二维凝胶上分离的多肽的结构信息。然而，蛋白质分析的最终目标必须是重建活细胞或器官中的蛋白质功能。为此，我们一直在使用不含变性剂的2-D PAGE。在本研究项目中，我们提出了四种二维PAGE技术(Type-I、Type-II、Type-III和Type-IV)相结合的方法，将由Type-IV二维PAGE获得的多肽结构信息与由Type-I二维PAGE获得的蛋白质功能信息相关联，进一步发展了新的蛋白质分离和回收的电泳技术，克服了传统的二维PAGE TE…的不足更多的小蛋糕。在传统的2-D PAGE中，将蛋白质电泳固定在平板凝胶上，用染料或硝酸银染色，通过安装在微型计算机中的图像分析软件进行定量，然后从凝胶片段中提取以进行进一步的结构分析。这些程序大多需要人工操作，而且几乎不可能实现自动化。在1996-1998年间，我们开发了用于分离蛋白质的毛细管电泳法。毛细管电泳的特点是柱上检测、柱上定量和分离过程的全自动化。建立了毛细管等电聚焦和毛细管十二烷基硫酸钠电泳法的分离条件，并将其用于人血浆蛋白的分离。然后，我们对毛细管电泳法的条件进行了扩展，旨在建立高效的蛋白质分离和回收技术。我们已经为此目的建造了几个装置和装置，我们正在评估它们在分离人血浆中次要蛋白质组分方面的性能。较少