Multi-angle light scattering and dynamic light scattering coupled to size exclusion chromatography

多角度光散射和动态光散射与尺寸排阻色谱联用

• 批准号: 494979009
• 金额: --
• 依托单位: Universität Ulm
• 依托单位国家: 德国
• 项目类别: Major Research Instrumentation
• 财政年份: 2021
• 资助国家: 德国
• 起止时间: 2020-12-31 至 无数据
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/494979009?language=en
• 关键词: Multi; angle; light; scattering; dynamic

Experiments in protein biochemistry and structural biology entirely rely on the availability of purified proteins in isolation or in complex with other proteins, RNA, DNA or lipids. Protein particle solutions are characterized by a number of different parameters like particle concentration, hydrodynamic radius and molar mass. The determination of these parameters and thus the indispensable information about the homogeneity of protein(complex) particles in solution is of utmost importance for understanding principles of their assembly and/or biogenesis and molecular functions. Furthermore, many downstream methods like crystal structure determination or biochemical and biophysical approaches such as in vitro-reconstitution experiments either depend on or at least benefit from such information.With this proposal, we apply for a setup consisting of a combination of multi-angle light scattering (determination of the absolute molar mass), dynamic light scattering (determination of hydrodynamic radius) and a refractive index detector (absorption independent concentration determination) coupled to size exclusion chromatography in a chromatography refrigerator. This setup will be used by several independent institutes and research groups at the Ulm University working with proteins or protein complexes, ensuring efficient and maximum utilization of the setup.The equipment will be used to determine the absolute molar mass as well as its distribution and hydrodynamic radii of native and unlabeled biological particles in solution. These particles may be proteins and protein-conjugate complexes (the conjugates being carbohydrates, nucleic acids or lipids). This information will help us to understand the molecular functions of biomolecules either in their healthy or diseased states or when used for therapeutic approaches. In addition, it also serves as tool for quality control in a number of projects and will significantly increase the chances of crystallization of proteins and their complexes. Since no comparable equipment is available on the entire campus, this equipment will considerably increase the analytical possibilities for biological samples at the Ulm University, including research conducted as part of three protein-centered, DFG-funded research networks on campus.

蛋白质生物化学和结构生物学的实验完全依赖于纯化蛋白质的分离或与其他蛋白质，RNA，DNA或脂质的复合物的可用性。蛋白质颗粒溶液的特征在于许多不同的参数，如颗粒浓度，流体动力学半径和摩尔质量。这些参数的确定，从而在溶液中的蛋白质（复合物）颗粒的均匀性的不可或缺的信息是最重要的了解其组装和/或生物起源和分子功能的原则。此外，许多下游方法，如晶体结构测定或生物化学和生物物理方法，如体外重构实验，依赖于或至少受益于这些信息。（绝对摩尔质量的测定），动态光散射（流体动力学半径的测定）和折射率检测器（吸收独立的浓度测定）与色谱冰箱中的尺寸排阻色谱偶联。该装置将被乌尔姆大学的几个独立研究所和研究小组用于蛋白质或蛋白质复合物的研究，确保装置的有效和最大限度的利用。该设备将用于确定溶液中天然和未标记生物颗粒的绝对摩尔质量及其分布和流体动力学半径。这些颗粒可以是蛋白质和蛋白质-缀合物复合物（缀合物是碳水化合物、核酸或脂质）。这些信息将帮助我们了解生物分子在健康或疾病状态下或用于治疗方法时的分子功能。此外，它还可作为许多项目的质量控制工具，并将显着增加蛋白质及其复合物结晶的机会。由于整个校园没有类似的设备，该设备将大大增加乌尔姆大学生物样本的分析可能性，包括作为三个以蛋白质为中心的DFG资助的校园研究网络的一部分进行的研究。