Simple Nuclei Preparation and Co-immunoprecipitation Procedures for Studying Protein Abundance and Interactions in Plant Circadian Time Courses.
Simple Nuclei Preparation and Co-immunoprecipitation Procedures for Studying Protein Abundance and Interactions in Plant Circadian Time Courses.
复制标题
DOI：
10.1007/978-1-0716-1912-4_11
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
植物生物钟调节在特定时间和季节发生的多种发育和生理事件。由于目前已知的许多时钟蛋白和时钟相关调节因子都是转录因子，因此分析细胞核中的分子事件至关重要。此外，经常需要对蛋白质丰度和相互作用进行长期过程分析，以评估生物钟对时钟调节现象的作用。在这里，我们介绍了一个简单的程序来制备核富集组织，我们通常使用它来研究低丰度核蛋白（即，转录因子）。除了测量丰度变化外，植物时间生物学研究还需要研究一天中特定时间或某些环境条件下蛋白质-蛋白质相互作用的动态变化。因此，我们还提出了我们的免疫共沉淀方法研究昼夜/昼夜蛋白质-蛋白质相互作用，在拟南芥和烟草核定位的蛋白质。
The plant circadian clock regulates multiple developmental and physiological events that occur at specific times and seasons. As many of the currently known clock proteins and clock-associated regulators are transcription factors, analyzing molecular events in the nuclei is crucial. In addition, long-time course analyses of protein abundance and interactions are often required to assess the role of the circadian clock on clock-regulated phenomena. Here we introduce a simple procedure to prepare nuclear-enriched tissues, which we routinely use to study time-resolved accumulation changes in low-abundance nuclear proteins (i.e., transcription factors). In addition to measuring changes in abundance, investigating the protein-protein interaction dynamics at specific times of day or under certain environmental conditions is needed for plant chronobiology studies. Therefore, we also present our co-immunoprecipitation method for studying diurnal/circadian protein-protein interactions, tailored to nuclear-localized proteins in Arabidopsis and tobacco.