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Author:
Date:
2014-08-05
[Abstract] Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are quenched when exposed to aqueous solution. The change in fluorescence can be used to follow unfolding of proteins induced by temperature, pH, or chaotropic agents. The method is well characterized for monomeric proteins. Here, we extend the application to supramolecular protein and nucleo-protein complexes using virus particles as an example. SYPRO-orange™ dye is the dye of choice because it is matched for use with q-PCR instruments and the ...
[摘要] 差示扫描荧光测定（DSF）是一种快速，经济，直接的技术，用于估计蛋白质的热稳定性。该原理涉及荧光染料与蛋白质的热暴露的疏水口袋的结合。在该技术中使用的染料在非极性环境中是高度荧光的，并且当暴露于水溶液时淬灭。荧光的变化可用于跟踪由温度，pH或离液剂诱导的蛋白质的解折叠。该方法充分表征了单体蛋白质。在这里，我们将应用程序扩展到超分子蛋白和核蛋白复合物使用病毒颗粒为例。 SYPRO-orange™染料是首选的染料，因为它与q-PCR仪器配合使用，荧光响应在宽的pH和温度范围内是稳定的。该技术相对于标准生物物理方法的优点包括对生物和技术重复的高通量筛选的能力和高灵敏度。
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