Simple Nuclei Preparation and Co-immunoprecipitation Procedures for Studying Protein Abundance and Interactions in Plant Circadian Time Courses.

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.