The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization.
The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization.
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DOI：
10.1002/0471142727.mb1028s94
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发表时间：
2011-04-01
影响因子：
--
通讯作者：
de la Pena, Andres Hernandez
中科院分区：
文献类型：
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作者：
Phillips, Kevin;de la Pena, Andres Hernandez
Thermofluor测定，也称为热位移测定或差示扫描荧光（DSF），是一种快速简单的方法，其基于蛋白质稳定性的高通量测量。Thermofluor方法可以在几乎所有的qPCR机器上进行，这意味着不需要特殊的仪器，并且可以用于验证蛋白质制剂的质量，筛选配体或辅因子，并发现最大化蛋白质稳定性的缓冲液和添加剂。本单元介绍了如何在几种常见型号的qPCR仪器上设置Thermofluor方法，如何制备用于测定的样品，以及如何运行和分析所得数据。该单元还描述了一个96孔筛选，以确定蛋白质稳定性的最佳缓冲液条件，这可能有助于蛋白质结晶，并详细介绍了定制软件的使用，专门用于分析来自Thermofluor筛选的数据。
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.