Phillips Kevin, de la Peña Andres Hernandez
The Methodist Hospital Research Institute, Houston, Texas, USA.
Curr Protoc Mol Biol. 2011 Apr;Chapter 10:Unit10.28. doi: 10.1002/0471142727.mb1028s94.
The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer conditions for protein stability, which may assist in protein crystallization, and details the use of custom software, developed specifically for the analysis of data from Thermofluor screens.
热荧光分析，也称为热迁移分析或差示扫描荧光法（DSF），是一种基于蛋白质稳定性高通量测量的快速简便方法。热荧光法几乎可以在所有定量PCR仪上进行，这意味着无需特殊仪器，并且可用于验证蛋白质制剂的质量、筛选配体或辅因子，以及发现能使蛋白质稳定性最大化的缓冲液和添加剂。本单元介绍了如何在几种常见型号的定量PCR仪上设置热荧光法，如何为该分析制备样品，以及如何运行和分析所得数据。本单元还介绍了一种用于确定蛋白质稳定性最佳缓冲条件的96孔筛选方法，这可能有助于蛋白质结晶，并详细说明了专门为分析热荧光筛选数据而开发的定制软件的使用方法。
