Fulneček Jaroslav, Říha Karel
Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.
Methods Mol Biol. 2021;2209:109-117. doi: 10.1007/978-1-0716-0935-4_7.
Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators.
基因表达所涉及的分子过程包含蛋白质与核酸之间的众多相互作用。对这些相互作用进行定量描述对于阐明转录、基因组复制和翻译的调控机制至关重要。在此，我们描述了一种基于蛋白质诱导荧光增强（PIFE）的蛋白质 - 核酸相互作用定量分析的详细方案。虽然PIFE主要用于单分子研究，但我们对其进行了改进，以便使用标准荧光酶标仪在微孔板中对蛋白质 - 核酸相互作用进行批量测量。微孔板PIFE测定法（mwPIFE）操作简单，无需费力地对蛋白质进行标记，且具有高通量。这些特性使mwPIFE成为需要进行多次平行测量的常规应用（如缓冲液优化、竞争实验或筛选结合调节剂的化学文库）的首选方法。
