MOLECULAR ASSEMBLY IN FLOW CYTOMETRY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。阐明大量新测序的蛋白质的功能是生物医学研究人员面临的下一个主要挑战，这些蛋白质是许多成功的基因组计划的产物。使这个问题复杂化的是，除了简单的数值问题之外，许多（如果不是大多数的话）蛋白质在大分子复合物中是活跃的。因此，了解这些复合物的组装是至关重要的。流式细胞术提供多参数检测，具有高灵敏度，优异的动力学分辨率和均匀的分析格式，这些特点已被用于研究许多分子组装在过去。然而，这些研究揭示了一些限制流式细胞术用于研究低亲和力分子组装的局限性，包括游离与结合的差区分，蛋白质与微球的非特异性和差结合，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，使分析和筛选低亲和力的分子组件。因此，我们将利用改进的激发光学，样品传递和数据采集来创建流式细胞仪，以提高游离与结合的仪器分辨率。我们还将评估由不同材料制成的微球对减少非特异性结合的作用，并开发改进的蛋白质附着方法。将我们以前开发的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，结合之前的发展，如第二代快速动态流式细胞仪，将在三个合作领域进行演示和测试，细胞分子组装，基于微球的分子组装和流动蛋白质组学。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合的研究和筛选中的实用性。

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.