CORE C: PROTEIN EXPRESSION AND CELL MORPHOLOGY

核心 C：蛋白质表达和细胞形态

• 批准号: 8360523
• 负责人: Sean M Ward
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360523
• 关键词: 3-Dimensional; Antibodies; Area; Binding Sites; Cell Culture Techniques; Cells; Cellular Morphology; Centers of Research Excellence; Complex; Confocal Microscopy; Cryoelectron Microscopy; Cultured Cells; Data; Detection; Disease; Electron Microscopy; Fluorescence Microscopy; Funding; Grant; Hypertrophy; Image; Imagery; Immunofluorescence Immunologic; Immunoprecipitation; Individual; Label; Laboratories; Measurement; Microfilaments; Mus; Muscle; Myosin Light Chain Kinase; National Center for Research Resources; Negative Staining; Nevada; Nomarski Interference Contrast Microscopy; Non-Insulin-Dependent Diabetes Mellitus; Phase; Phenotype; Phosphoric Monoester Hydrolases; Population Heterogeneity; Preparation; Principal Investigator; Protein Overexpression; Proteins; Proteomics; Protocols documentation; Research; Research Infrastructure; Resources; Sampling; Services; Site; Smooth Muscle; Smooth Muscle Myocytes; Sorting - Cell Movement; Source; Structure; Support System; Tissues; Transfection; United States National Institutes of Health; Visceral; Western Blotting; citrate carrier; cost; digital; gel electrophoresis; light microscopy; protein expression; reconstruction; sample fixation; tissue preparation

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 使用蛋白质分析结合多种形态学方法来检查蛋白质表达的服务，以定位具有异质细胞群的组织中的表达水平。核心集成了西方分析和形态学研究，从基本形态学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中进行，为各个项目提供无缝支持机制。核心提供的服务包括蛋白过表达、组织和细胞中的蛋白表达分析、转染 细胞、细胞培养、细胞裂解物制备、免疫沉淀、组织制备、蛋白质印迹、凝胶电泳、nanoLC 和 MALD1 样品制备。该核心还将充当与内华达蛋白质组学核心的接口，用于质谱分析和 MALD1 数据分析。该支持系统集成了组织的结构分析以及组织和分离细胞内蛋白质表达位点的可视化。将在光学显微镜（明场、相差和微分干涉显微镜）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）领域的形态学专业知识，用于检查包含多种细胞表型的异质组织中的蛋白质表达。核心将解决抗体的使用问题（即固定方案、非特异性标记），以优化蛋白质的使用和检测。数字重建将进行去卷积和 3 维体积 需要时进行重建 该核心还将为在从 smMHCICreleGFP 小鼠中分选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。该核心还将提供肌肉和孤立的超微结构分析方面的专业知识。 肌丝。将支持多个专业领域，包括传统电子显微镜、分离肌丝负染色、冷冻电子显微镜和蛋白质免疫金标记。超微结构分析对于确定平滑肌中发生的病理变化是必要的 由于 II 型糖尿病等疾病导致的细胞肥大和结构变化。将使用负染色和免疫金标记对肌动蛋白丝进行分析，以确定肌球蛋白轻链激酶和 磷酸酶。