GST Pull-Down Assay to Study PIF4 Binding In Vitro

The phytochrome-interacting factor 4 (PIF4) is a well-known transcription factor that plays a pivotal role in plant thermomorphogenesis, coordinating growth and development in response to temperature changes. As PIF4 functions by forming complexes with other proteins, determining its interacting partners is essential for understanding its diverse roles in plant thermal responses. The GST (glutathione-S-transferase) pull-down assay is a widely used biochemical technique that enables the investigation of protein-protein interactions in vitro. It is particularly useful for studying transient or weak interactions between proteins. In this chapter, we describe the GST pull-down approach to detect the interaction between PIF4 and a known or suspected interacting protein. We provide detailed step-by-step descriptions of the assay procedures, from the preparation of recombinant GST-PIF4 fusion protein to the binding and elution of interacting partners. Additionally, we provide guidelines for data interpretation, quantification, and statistical analysis to ensure robust and reliable results.