Fluorescence Polarization Assay to Quantify Protein-Protein Interactions in an HTS Format
Fluorescence Polarization Assay to Quantify Protein-Protein Interactions in an HTS Format
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DOI：
10.1007/978-1-4939-2425-7_35
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发表时间：
2015-01-01
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影响因子：
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通讯作者：
Du, Yuhong
中科院分区：
文献类型：
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作者：
Du, Yuhong
荧光偏振（FP）技术是以测量分子旋光度为基础的一种技术，已被广泛应用于溶液中分子间相互作用的研究。如果结合分子中的一个相对较小且具有荧光，则该方法可用于测量两个分子之间的结合和解离。荧光标记的小分子（如小肽）在溶液中快速旋转。在被偏振光激发时，发射的光保持去偏振并产生低FP信号。当溶液中的荧光小分子与更大的分子（如蛋白质）结合时，复合物的运动变得更慢。当这种复合物被偏振光激发时，由于复合物的缓慢运动，大部分发射的光都是偏振的。因此，荧光标记的小分子与较大分子的结合可以通过偏振的变化来监测，并通过产生增加的FP信号来测量。本章旨在提供一个逐步的实用程序，用于开发多孔板格式的FP检测试剂盒，以监测同质格式的蛋白质-蛋白质相互作用（PPI）。
Fluorescence polarization (FP) technology is based on the measurement of molecule rotation, and has been widely used to study molecular interactions in solution. This method can be used to measure binding and dissociation between two molecules if one of the binding molecules is relatively small and fluorescent. The fluorescendy labeled small molecule (such as a small peptide) rotates rapidly in the solution. Upon excitation by polarized light, the emitted light remains depolarized and gives rise to a low FP signal. When the fluorescent small molecules in solution are bound to bigger molecules (such as a protein), the movement of the complex becomes slower. When such a complex is excited with polarized light, much of the emitted light is polarized because of the slow movement of the complex. Thus, the binding of a fluorescendy labeled small molecule to a bigger molecule can be monitored by the change in polarization and measured by the generation of an increased FP signal. This chapter aims to provide a step-by-step practical procedure for developing an FP assay in a multi-well plate format to monitor protein-protein interaction (PPI) in a homogenous format.