Self-Assembling Autofluorescent Protein Microarrays, a Universal Resource for the Plant Research Community

项目描述。该项目由亚利桑那大学的四位研究人员David Galbraith、David Gang、Paul Haynes和Serrine Lau合作完成。他们将共同开发新的高通量技术，用于真核生物中蛋白质及其相互作用的综合分析。该项目包括一系列概念验证实验，主要使用拟南芥和玉米作为实验生物。该项目基于最近在项目实验室实施的描述，即通过原位生物合成过程生产蛋白质微阵列。这包括在显微镜载玻片上发现不同的重组DNA结构，这些结构编码感兴趣的蛋白质融合到表位标签上。与DNA混合并同时被发现的是一种针对抗原表位的抗体。在定位和固定混合DNA/抗体混合物后，将微阵列覆盖在转录/翻译混合物中，该混合物从DNA分子中编码的信息产生表位标记蛋白，然后被固定的抗表位抗体捕获。由此产生的蛋白质微阵列将用于各种新的下游分析，这些分析已在参与实验室中设计，用于检查蛋白质-蛋白质相互作用，蛋白质- dna相互作用，蛋白质元件的共价修饰以及蛋白质元件的质谱表征。这些检测将通过明智地选择阳性和阴性对照来验证，并将扩展到解决社区感兴趣的特定生物学问题，从而使社区了解这些技术的可用性和力量。预期的结果。该项目的预期结果有三个方面：分子工具包（试剂，重组DNA分子，蛋白质编码微阵列），如何使用该工具包进行体外蛋白质相互作用检查的相关描述，以及为验证方法而进行的实验结果。所有结果将在验证和/或发表后的三个月内从加尔布雷斯博士的实验室自由地传播给科学界，以先到者为准。分子工具包将按要求提供给感兴趣的个人。适度的收费将被实施，以收回常规生产常用试剂和微阵列的成本。作为该项目的一部分，重组DNA分子将不收费。方法描述和实验结果将发布在项目网站上，并酌情在科学文献中发表。这些方法的设计在范围上是完全通用的，并且应该可以转移到其他真核生物，包括植物界以外的真核生物。项目网站为：http://cals.arizona.edu/research/HTPM

Project Description. This project represents a collaboration between four investigators at the University of Arizona, David Galbraith, David Gang, Paul Haynes, and Serrine Lau. Together they will develop novel, high throughput techniques for comprehensive analysis of proteins and their interactions within eukaryotic organisms. The project comprises a series of proof-of-concept experiments primarily using Arabidopsis thaliana and Zea mays as the experimental organisms. The project is based on the recent description, which has been implemented in the project laboratories, of the production of protein microarrays via a process of in situ biosynthesis. This involves spotting onto microscope slides different recombinant DNA constructions encoding the proteins of interest fused to an epitope tag. Mixed with the DNA, and spotted at the same time, is an antibody directed against the epitope. After spotting and immobilizing the mixed DNA/antibody mixture, the microarrays are covered in transcription/translation mix, which produces epitope-tagged proteins from the information encoded in the DNA molecules, which are then captured by the immobilized anti-epitope antibodies. The resultant protein microarrays will then be employed for a variety of novel downstream assays, which have been devised in the participating laboratories to examine protein-protein interactions, protein-DNA interactions, covalent modification of protein elements, and mass-spectrometric characterization of the protein elements. These assays will be validated by judicious choice of positive and negative controls, and will be extended to address specific biological questions of interest to the community thereby informing the community of the availability and power of these techniques. Expected Outcomes. The expected outcomes of this project are three-fold: a molecular toolkit (reagents, recombinant DNA molecules, protein-encoding microarrays), the associated descriptions of how to use this toolkit for examination of protein interactions in vitro, and the results of the experiments that have been done to validate the methodology. All outcomes will be freely disseminated from the laboratory of Dr. Galbraith to the scientific community within three months of their validation and/or publication whichever comes first. The molecular toolkits will be provided to interested individuals on request. Modest fees will be implemented to recover costs of routine production of popular reagents and microarrays. No charges will be made for recombinant DNA molecules produced as part of the project. The descriptions of the methods and the results from the experiments will be posted to the project website and, as appropriate, will be published in the scientific literature. The methods are designed to be entirely general in scope, and should be transferable to other eukaryotic organisms, including those of other than the plant kingdom. The project website is: http://cals.arizona.edu/research/HTPM