CORE C: PROTEIN EXPRESSION AND CELL MORPHOLOGY

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 使用蛋白质分析结合各种形态方法来定位具有不同细胞群体的组织中的表达水平的蛋白质表达检查服务。核心整合了西方分析和形态研究，从基本形态到复杂的三维体积测量或电子显微镜的定量蛋白质表达和成像需求将在同一实验室进行，为个别项目提供无缝的支持机制。Core将提供的服务包括蛋白质过表达、组织和细胞中的蛋白质表达分析、 细胞，细胞培养，细胞裂解制备，免疫沉淀，组织制备，蛋白质印迹，凝胶电泳，纳米LC和MALD1样品制备。该核心还将作为与内华达蛋白质组学核心的接口，以进行质谱分析和MALD1数据分析。在这个支持系统中集成了对组织的结构分析以及组织和分离细胞内蛋白质表达位置的可视化。将在光学显微镜(明视场、相差显微镜和差分干涉对比显微镜)领域为拟议的内脏组织组织学研究提供支持。该核心还将提供免疫荧光(常规荧光和共聚焦显微镜)领域的形态学专门知识，用于检查包含几种细胞表型的不同组织中的蛋白质表达。该中心将对抗体的使用(即固定方案、非特异性标记)进行故障排除，以优化蛋白质的使用和检测。数字重建将被去卷积和三维体积 在需要时进行的重建核心还将为在分离smMHCICeleGFP小鼠的平滑肌细胞之前检查组织和分离细胞中的EGFP表达提供支持。核心还将提供肌肉和分离的超微结构分析方面的专业知识 肌丝。将支持几个专业领域，包括常规电子显微镜、分离肌丝的阴性染色、冷冻电子显微镜和蛋白质的免疫金标记法。有必要进行超微结构分析以确定发生在平滑肌上的病理变化 细胞在肥大过程中，以及由于II型糖尿病等疾病而引起的结构变化。使用负染色和免疫金标记法对肌动蛋白细丝进行分析，以确定肌球蛋白轻链激酶和 磷酸酶。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. service for the examination of protein expression using western analysis combined with a variety of morphological approaches to localize levels of expression in tissues with heterogeneous populations of cells. The Core has integrated western analysis and morphological studies such that quantitative protein expression and imaging needs from basic morphological to complex 3-dimensional volumetric measurements or electron microscopy will be performed in the same laboratory providing a seamless support mechanism for individual projects. The services to be provided by the Core include protein overexpression, protein expression analysis in tissues and cells, transfections of cells, cell culture, cell lysate preparations, immunoprecipitations, tissue preparations, western blots, gel electrophoresis, nanoLC and MALDl sample preparation. The Core will also act as an interface with the Nevada Proteomics Core for mass spectral analysis and analysis of MALDl data. Integrated within this support system with be a structural analysis of tissues and visualization of the sites of protein expression within tissues and isolated cells. Support will be provided in the areas of light microscopy (bright field, phase contrast and differential interference contrast microscopy) for the proposed histological studies on visceral tissues. The Core will also provide morphological expertise in the areas of immunofluorescence (regular fluorescence and confocal microscopy) for the examination of protein expression in heterogeneous tissues that contain several cellular phenotypes. The Core will troubleshoot the use of antibodies (i.e. fixation protocols, non-specific labeling) to optimize use and the detection of proteins. Digital reconstructions will be deconvolved and 3-dimensional volume reconstructions performed when needed The core will also provide support for the examination of eGFP expression in tissues and isolated cells prior to the sorting of smooth muscle cells from smMHCICreleGFP mice. The Core will also provide expertise in the ultrastructural analysis of muscles and isolated myofilaments. Several areas of expertise will be supported including conventional electron microscopy, negative staining of isolated myofilaments, cryoelectron microscopy and immunogold labeling of proteins. Ultrastructural analysis is necessary to determine the pathological changes that occur in smooth muscle cells during hypertrophy and changes in structure due to diseases like type II DM. Analysis of actin filaments using negative staining and immunogold labeling will be provided to determine the binding sites of myosin light chain kinase and phosphatase.