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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 我们正在使用可克隆的标签（金属沉淀肽和金属硫蛋白），以识别，定位和研究EM中的蛋白质。 这些可插的标签将扮演与GFP在光学显微镜中发挥的作用相似的作用。 我们目前正在使用的标签是A3（金质肽），GE8（银色沉淀肽）和金属硫硫蛋白，MTH（一种重金属结合蛋白）。 在我们的实验室中研究良好的蛋白质被用作我们实验的载体。 EG5（动力蛋白运动结构域），KAR3（动力蛋白运动结构域），波形蛋白（中间丝蛋白），脱氨蛋白（另一种中间丝蛋白）和VP1（多瘤病毒的主要衣壳蛋白） 制作的结构包括： EG5+A3，EG5+GE8，EG5+3A3，EG5+2mths Kar3+A3，Kar3+2mths 波形蛋白+A3，波形蛋白+2mths，波形蛋白+1mth Desmin+1mth VP1+A3，VP1+GE8，VP1+3A3 在我们通过肽标签优化金属沉淀和结合反应的努力中，我们进行了一系列反复试验实验。 这些实验涉及通过多种方法制备样品，使用各种浓度和类型的黄金前体，不同浓度的蛋白质以及不同的浓度和减少缓冲液的类型。 经过大量的实验，我们将精力集中在构建体上EG5+A3，Vimentin+A3，Kar3+A3，Vimentin+A3，Desmin+1mth+1mth+1mth+1mmth，这是根据我们的初步数据显示的。