Analytical Ultracentrifugation for Complex Systems

申请人描述：描述两国之间的相互作用 分子是我们理解正常和病理的核心。 生物化学。因此，对分子相互作用的准确描述 在开发安全有效的药物方面起着至关重要的作用。 分子表征仪器和方法的改进 互动对于研究人员有效地推进基础医学是必要的 科学。分析超速离心法是世界上最严谨、最强大的 和适用的方法确定的强度，化学计量比和大小 分子络合物，但目前仅限于相对分子的分析 简单的生化系统。随着进一步的发展，分析 超速离心法将适用于复杂得多的问题 而不是用目前的技术所能研究的。总体目标是带来 分析超速离心法处理问题的力量和严谨性 对分子和细胞生物学感兴趣。合作努力一直是 与美国国立卫生研究院资助的几名研究人员建立，他们使用分析 在他们的工作中使用超速离心法，并需要由 这项提议的三个具体目标： 1)快速扫描吸收光学的发展。一种基于光电二极管阵列的 能够提供10微米S体内浓度分布的探测器 将开发径向分辨率。波长选择需要&lt；1 S 重现性优于+/-1 nm。这些改进将使 多波长快速沉积边界的分析。 2)放射性标记和免疫化学发光探测器的研制。细胞 将开发窗口和中间件以允许使用发光 用于放射性标记和免疫化学发光检测的检测器。它的特殊性 灵敏度将允许在条件下对分子进行表征 接近体内发现的那些。新的中心摆件将减少样品数量 体积增加多达40倍，同时增加每次实验的样本数 高达4倍，使其能够快速有效地进行 实验方案涉及大量变量。 3)一体化超速离心机操作系统的开发。一种新的 将开发仪器操作系统，使仪器更容易 从逻辑上操作并提高数据收集和分析的效率 组织数据采集程序。

APPLICANT'S DESCRIPTION: The characterization of the interactions between molecules is central to our understanding of both normal and pathological biochemistry. Consequently, the accurate description of molecular interactions plays a critical role in the development of safe and effective drugs. Improvements in the instruments and methods for characterizing molecular interactions are necessary for researchers to efficiently advance basic medical science. Analytical ultracentrifugation is one of the most rigorous, powerful and adaptable methods for determining the strength, stoichiometry and size of molecular complexes, but is currently limited to the analysis of relatively simple biochemical systems. With further development, analytical ultracentrifugation would be applicable to problems that are far more complex than can be investigated using current technology. The overall goal is to bring the power and rigor of analytical ultracentrifugation to bear on problems of interest in molecular and cellular biology. Collaborative efforts have been established with several NIH-funded researchers who use analytical ultracentrifugation in their work and who require the advances provided by the three specific aims of this proposal: 1) Development of rapid-scan absorbance optics. A photodiode-array-based detector capable of providing concentration profiles in < 1 s and at a 10 um radial resolution will be developed. Wavelength selection will require < 1 S with a reproducibility better than +/- 1 nm. These improvements will allow the analysis of rapidly-sedimenting boundaries at multiple wavelengths. 2) Development of radiolabel and Immuno-chemiluminescence detectors. Cell windows and centerpieces will be developed to permit the use of a luminescence detector for radiolabel and immuno-chemiluminescence detection. The specificity and sensitivity will allow the characterization of molecules under conditions approaching those found in vivo. The new centerpieces will decrease sample volume by up to 40-fold, while increasing the number of samples per experiment up to 4-fold, making it possible to rapidly and effectively conduct experimental protocols involving a large number of variables. 3) Development of an integrated ultracentrifuge operating system. A new instrument operating system will be developed to make the instrument easier to operate and improve the efficiency of data collection and analysis by logically organizing data acquisition procedures.