CORE--LIPOPROTEIN ANALYSES

核心——脂蛋白分析

基本信息

批准号：
6202168
负责人：
PATRICIA BLANCHE
金额：
$ 35.89万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-30 至 2000-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6202168
关键词：
apolipoproteins atherosclerosis biomedical facility blood lipoprotein blood lipoprotein metabolism enzyme linked immunosorbent assay gas chromatography gel electrophoresis immunoaffinity chromatography immunochemistry immunologic assay /test lipid structure ultracentrifugation western blottings

项目摘要

Core A will provide standardized measurements of lipids, lipoproteins, lipoprotein subclasses and apoproteins in plasma and lipoprotein subfractions on mice used in research project studies. Immunochemical support of these studies will include development of apoprotein and enzyme specific immunoassays and immunoaffinity separation procedures. Specialize ultracentrifugation and gel filtration procedures will be performed for isolation of lipoproteins subspecies. Function: 1. Lipid analyses - Lipid concentrations of individual lipds will be determined by standardized, micro-scale enzyme-endpoint assay methods and gas-lipid chromatography (GLC). 2. Lipoprotein analyses - Whole plasma and lipoprotein fractions will be analyzed by nondenaturing gradient gel electrophoresis and computer-based densitometry. Plasma lipoprotein fractions and subfractions will be obtained within standard ultracentrifugal density intervals and by gel filtration techniques. Plasma lipoproteins will be separated by electrophoretic mobility on agarose gels followed by lipid staining and/or determination of apoprotein associations using Western blotting procedures. Gel filtration will be performed on mouse plasma to obtain fractions for analysis of lipid, apoprotein and enzyme concentrations. 3. Apoprotein analyses - Quantification of apoproteins B, CIII, AI, AII, E, Lp(a), and albumin will be performed using standardized enzyme-linked immunoassay (ELISA). Apoprotein associations on the same lipoprotein particle will be quantified in plasma and isolated lipoprotein fractions using magnetized immunoaffinity beads for lipoprotein capture and routine ELISA measurements to identify associated apoproteins by difference from the total sample apoprotein measurement. This technique will be used to identify apoprotein-associated enzyme activity and mass levels, as well. 4. Immunoassay and research support - In support of the immunochemical procedures, purified apoprotein and apoprotein- specific antisera stocks will be prepared and characterized for use in immunoassays and immunoaffinity separation procedures, and will be available to research projects for Western and dot blotting procedures. ELISA quantitation of human and mouse apoproteins, enzymes, and proteins of interest to research projects will be standardized in accordance with research project requirements. Standardized and quality controlled non-denaturing gradients gels in both 2-14% and 3- 31% acrylamide concentrations will be provided to research projects as needed. The Core A centrifugation facility will provide maintenance and repair of ultracentrifuges, centrifuge rotors, and training in standard and specialized density separations. Databases for storage and retrieval of analytic data will be maintained.

核心A将提供脂质，脂蛋白的标准化测量值血浆和脂蛋白中的脂蛋白亚类和顶蛋白研究项目研究中使用的小鼠的子量。这些研究的免疫化学支持将包括开发载脂蛋白和酶特异性免疫测定和免疫接触分离程序。专门为超速离心和凝胶过滤将进行分离脂蛋白亚种的程序。功能：1。脂质分析 - 单个脂质的脂质浓度将通过标准化的微尺度酶 - 端点测定确定方法和气脂色谱法（GLC）。 2。脂蛋白分析 - 整个血浆和脂蛋白分数将由非定位梯度凝胶电泳和基于计算机的光密度法。血浆脂蛋白级分和子部分将是在标准的超速离心密度间隔内获得过滤技术。血浆脂蛋白将通过琼脂糖凝胶上的电泳迁移率，然后进行脂质染色和/或使用蛋白质印迹确定载脂蛋白缔合程序。凝胶过滤将在小鼠血浆上进行获得分数以分析脂质，载脂蛋白和酶浓度。 3。载脂蛋白分析 - 定量载脂蛋白B，CIII，AI，AII，E，LP（A）和白蛋白将进行使用标准化酶联免疫测定（ELISA）。载蛋白同一脂蛋白颗粒上的关联将在使用磁化的等离子体和分离的脂蛋白分数用于脂蛋白捕获和常规ELISA的免疫亲和力珠通过与总样品载蛋白测量。将使用此技术确定载毒素相关的酶活性和质量水平，为出色地。 4。免疫分析和研究支持 - 支持免疫化学程序，纯化的载脂蛋白和载脂蛋白将准备特定的反塞库存，并以此为特征免疫测定和免疫亲和力分离程序，将是可用于研究西方和DOT印迹程序的项目。 ELISA定量人和小鼠股蛋白，酶以及研究项目感兴趣的蛋白质将在根据研究项目要求。标准化和 2-14％和3-的质量控制的非降解梯度凝胶研究项目将提供31％的丙烯酰胺浓度需要。核心一个离心设施将提供维护并修复超速离心，离心转子和训练标准和专业密度分离。存储数据库并将检索分析数据。