TWO DIMENSIONAL ANALYTICAL PREPARATIVE ULTRACENTRIFUGE

描述：（改编自申请人摘要）本提案旨在 开发一个综合的分析-分析二维分区 超集中系统，基于在橡树岭开发的早期原型， 准确测量沉积速率和等密度线 细胞、亚细胞器和感染细胞的带密度（p） 试剂，并进行高分辨率的制备分离的基础上， 这些参数。 纯化的级分将通过高效液相色谱法分析。 分辨率二维电泳，目的是 为每种可以检测到的蛋白质分配特定的亚细胞 位置. 初步论证和可行性研究将在 涉及分离和分析不同类别的肝脏， 脑核团 LSB目前正在准备标准参考地图， 人类和啮齿类动物的蛋白质， 组成或序列，并绘制药物，激素， 和基因表达的毒性因子。 我们现在需要的方法 产生亚细胞部分（细胞核，线粒体，过氧化物酶体， 溶酶体等）可重复地，并且为了进一步细分这些 进入核膜、核仁、核孔复合体、内部和外部 线粒体膜等，这样每个蛋白质基因的位置 产品可以确定。 2D离心机系统还将 可用于检测和分离感染原， 组织和体液。

DESCRIPTION: (Adapted from the applicant's abstract) This proposal is to develop a combined preparative-analytical two-dimensional zonal ultracentrifuge system, based on early prototypes developed at Oak Ridge, to both measure accurately the sedimentation rates (s) and isopycnic banding densities (p) of cells, subcellular organelles and infectious agents, and to perform high resolution preparative separations based on these parameters. The purified fractions will be anaylyzed by high resolution two-dimensional electrophoresis with the objective of assigning to each protein that can be detected a specific subcellular location. Initial demonstration and feasibility studies will be concerned with separating and analyzing different classes of liver and brain nuclei. LSB is currently preparing standard reference maps of human and rodent proteins by organ, is identifying proteins by composition or sequence, and is mapping the effects of drugs, hormones, and toxic agents of gene expression by 2DE. We now require methods for producing subcellular fractions (nuclei, mitochondria, peroxisomes, lysosomes, etc.) reproducibly, and for further subfractionating these into nuclear membranes, nucleoli, nuclear pore complexes, inner and outer mitochondrial membranes, etc., so that the location of each protein gene product can be determined. The 2D centrifuge systems proposes will also be useful for the detection and isolation of infectious agents from tissues and body fluids.