NEW LABELING TECHOLOGIES FOR EM

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。 我们正在使用可克隆的标签(金属沉淀肽和金属硫蛋白)来识别、定位和研究EM中的蛋白质。这些可克隆的标签将发挥类似于GFP在光学显微镜中的作用。 我们目前正在研究的标签是A3(一种金沉淀肽)，Ge8(一种银沉淀肽)和金属硫蛋白，MTH(一种重金属结合蛋白)。 我们实验室研究得很好的蛋白质被用作我们实验的载体。EG5(运动区的运动区)、Kar3(运动区的运动区)、Vimentin(中间丝蛋白)、Desmin(另一种中间丝蛋白)和VP1(多瘤病毒的主要衣壳蛋白) 构建的构件包括： EG5+A3、EG5+Ge8、EG5+3A3、EG5+2个月 Kar3+A3，Kar3+2个月 波形蛋白+A3、波形蛋白+2个月、波形蛋白+1个月 Desmin+1个月 Vp1+A3、Vp1+Ge8、Vp1+3A3 在我们努力优化金属沉淀和多肽标记结合反应的过程中，我们进行了一系列反复试验。这些实验包括通过各种方法制备样品，使用不同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，我们正在集中精力构建EG5+A3、Vimentin+A3、Kar3+A3、Vimentin+A3、Desmin+1mth和Vimentin+1mMTH结构，这些结构基于我们的初步数据显示出最有希望的结构。

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.