MOLECULAR ASSEMBLY IN FLOW CYTOMETRY

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 生物医学研究人员面临的下一个主要挑战是阐明大量新测序蛋白质的功能，这些蛋白质是许多成功的基因组计划的产物。 除了简单的数字问题之外，使这个问题复杂化的是，许多（如果不是大多数）蛋白质在大分子复合物中具有活性。因此，理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相测定形式，这些特征在过去已被用于研究许多分子组装体。然而，这些研究已经揭示了阻止流式细胞术用于研究低亲和力分子组装体的几个限制，包括游离与结合的区分差、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，使低亲和力分子组装体的分析和筛选。因此，我们将创建一种流式细胞仪，利用改进的激发光学器件、样本输送和数据采集来提高自由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，以减少非特异性结合，并开发改进的蛋白质附着方法。 将我们先前开发的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，结合以前的发展，如第二代快速动力学流式细胞仪，将在三个合作领域，细胞分子组装，基于微球的分子组装和蛋白质组学的流动进行演示和测试。 总之，我们将显着提高和证明流式细胞术在低亲和力分子组装体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.