PROTEIN INTERACTION SCREENING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 阐明大量新测序的蛋白质的功能，这些蛋白质是许多成功基因组计划的产物，是生物医学研究人员面临的下一个主要挑战。除了简单的数字问题，使这个问题变得复杂的是，许多蛋白质(如果不是大多数的话)在大分子复合体中是活跃的。因此，了解这些复合体的组装是至关重要的。流式细胞术提供了高灵敏度、良好的动力学分辨率和均一的分析形式的多参数检测，这些特点在过去已经被用于许多分子组装的研究。然而，这些研究揭示了几个限制，阻碍了流式细胞术用于低亲和力分子组装的研究，包括对蛋白质与微球的游离态和结合态的辨别能力差，非特异性和结合能力差，以及有限的温度调节。该项目的目的是开发改进的方法和仪器，以实现对低亲和力分子组件的分析和筛选。因此，我们将利用改进的激发光学、样品传输和数据采集来创建一种流式细胞仪，以提高仪器对自由和束缚的分辨率。我们还将评估由不同材料创建的微球减少非特异性结合，并开发改进的蛋白质附着方法。将我们之前开发的Peltier单元技术扩展到仪器的整个样品路径，将增强温度调节。最后，上述技术与之前的发展，如第二代快速动力流式细胞仪，将在细胞分子组装、基于微球的分子组装和Flow中的蛋白质组学三个合作领域进行演示和测试。总之，我们将极大地改进和展示流式细胞术在低亲和力分子组件的研究和筛选中的应用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Elucidating the functions of the multitudes of newly sequenced proteins, which are the products of the many successful genome projects, is the next major challenge facing biomedical researchers. Complicating this problem, beyond the simple numerical issues, is the fact that many, if not most, proteins are active in large macromolecular complexes. Consequently, it is critical to understand the assembly of these complexes. Flow cytometry offers multiple parameter detection with high sensitivity, excellent kinetic resolution and a homogeneous assay format and these features have been used to study many molecular assemblies in the past. However, such studies have revealed several limitations that prevent flow cytometry from being used to study low affinity molecular assemblies, including poor discrimination of free vs. bound, nonspecific and poor binding of proteins to microspheres, and limited temperature regulation. The purpose of this project is to develop improved methods and instrumentation to enable analysis of and screening for low affinity molecular assemblies. Therefore we will create a flow cytometer utilizing improved excitation optics, sample delivery and data acquisition to enhance instrumental resolution of free vs. bound. We will also evaluate microspheres created of different materials for reduced nonspecific binding and develop improved protein attachment methods. Extending our previously developed Peltier unit technology to the entire sample path of the instrument will enhance temperature regulation. Finally, the above technologies, in conjunction with previous developments, such as the second generation rapid kinetic flow cytometer, will be demonstrated and tested in three collaborative areas, cellular molecular assembly, microsphere based molecular assembly and proteomics in flow. In summary, we will dramatically improve and demonstrate the utility of flow cytometry in the study and screening of low affinity molecular assemblies.