热位移分析筛选蛋白质稳定剂的缓冲液和添加剂：实用方法。
Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach.
机构信息
Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal.
UCIBIO, Applied Molecular Biosciences Unit, Chemistry Department, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal.
出版信息
Methods Mol Biol. 2023;2652:199-213. doi: 10.1007/978-1-0716-3147-8_11.
Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (T). One of its main applications is to identify the best buffers and additives that enhance protein stability.Understanding the TSA operating mode and the main methodological steps is a central key to designing effective experiments and retrieving meaningful conclusions. This chapter intends to present a straightforward TSA protocol, with different troubleshooting tips, to screen effective protein stabilizers such as buffers and additives, as well as data treatment and analysis. TSA results provide conditions in which the protein of interest is stable and therefore suitable to carry out further biophysical and structural characterization.
热迁移分析（TSA），也通常通过差示扫描荧光法（DSF）或 ThermoFluor 设计，是一种相对易于实施和执行的技术，在众多应用中非常有用。除了多功能性，它也非常便宜，因此适合高通量方法。TSA 使用荧光染料来监测研究中蛋白质的热变性，并确定其熔点（T）。其主要应用之一是确定最佳的缓冲液和添加剂，以增强蛋白质稳定性。了解 TSA 的工作模式和主要方法步骤是设计有效实验和得出有意义结论的关键。本章旨在介绍一种简单的 TSA 方案，包括不同的故障排除技巧，以筛选有效的蛋白质稳定剂，如缓冲液和添加剂，以及数据处理和分析。TSA 结果提供了感兴趣的蛋白质稳定的条件，因此适合进行进一步的生物物理和结构特征分析。
Zotero 插件