NEW LABELING TECHOLOGIES FOR EM

新兴市场的新标签技术

• 批准号: 8362561
• 负责人: DAN L. FELDHEIM
• 金额: $ 1.06万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362561
• 关键词: Binding; Binding Proteins; Buffers; Capsid Proteins; Cell physiology; Cellular Structures; Data; Desmin; Funding; Gold; Grant; Heavy Metals; Intermediate Filament Proteins; Kinesin; Label; Metallothionein; Metals; Methods; Motor; National Center for Research Resources; Peptides; Play; Polyomavirus; Precipitation; Principal Investigator; Proteins; Reaction; Research; Research Infrastructure; Resources; Role; Sampling; Series; Silver; Source; United States National Institutes of Health; Vimentin; Work; base; cost; light microscopy; research study

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. We are using clonable tags (metal precipitating peptides and Metallothionein) in our effort to identify, localize and study proteins in the EM. These clonable tags will play a role similar to what GFP plays in light microscopy. The tags we are currently working with are A3 (a gold precipitating peptide), Ge8 (a silver precipitating peptide) and Metallothionein, MTH (a heavy metal binding protein). Proteins well studied in our lab were used as a vehicle for our experiments. Eg5 (a kinesin motor domain), Kar3 (a kinesin motor domain), Vimentin (an intermediate filament protein), Desmin (another intermediate filament protein) and VP1 (a major capsid protein of the polyomavirus) Constructs made include: Eg5+A3, Eg5+Ge8, Eg5+3A3, Eg5+2MTHs Kar3+A3, Kar3+2MTHs Vimentin+A3, Vimentin+2MTHs, Vimentin+1MTH Desmin+1MTH VP1+A3, VP1+Ge8, VP1+3A3 In our efforts to optimize the metal precipitation and binding reactions by the peptide labels, we conducted a series of trial-and error experiments. These experiments involved preparing sample by a variety of methods, using various concentrations and types of gold precursors, different concentrations of proteins and also different concentrations and types of reducing buffers. After a large number of experiments, we are concentrating our efforts on constructs Eg5+A3, Vimentin+A3, Kar3+A3, Vimentin+A3, Desmin+1MTH and Vimentin+1mMTH which based on our preliminary data show the most promise.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们正在使用克隆标签（金属沉淀肽和金属硫蛋白）在我们的努力，以确定，定位和研究蛋白质的EM。 这些可克隆的标签将发挥类似于GFP在光学显微镜中所起的作用。 我们目前正在研究的标签是A3（一种金沉淀肽）、Ge 8（一种银沉淀肽）和金属硫蛋白、MTH（一种重金属结合蛋白）。 我们实验室研究的蛋白质被用作我们实验的载体。 Eg 5（驱动蛋白马达结构域）、Kar 3（驱动蛋白马达结构域）、波形蛋白（中间丝蛋白）、结蛋白（另一种中间丝蛋白）和VP 1（多瘤病毒的主要衣壳蛋白） 建造的建筑物包括： Eg 5 +A3、Eg 5 + Ge 8、Eg 5 + 3A 3、Eg 5 + 2个月 Kar 3 +A3，Kar 3 + 2个月 波形蛋白+A3、波形蛋白+2个月、波形蛋白+1个月 结蛋白+1MTH VP1+A3、VP1+ Ge 8、VP1+3A3 在我们的努力，以优化金属沉淀和结合反应的肽标记，我们进行了一系列的试错实验。 这些实验涉及通过各种方法制备样品，使用各种浓度和类型的金前体、不同浓度的蛋白质以及不同浓度和类型的还原缓冲液。 经过大量的实验，我们集中精力构建Eg 5 +A3、Vimentin+A3、Kar 3 +A3、Vimentin+A3、Desmin+1 MTH和Vimentin+1 mMTH，这些构建体基于我们的初步数据显示出最有希望。