Abstract
Protein-protein interactions (PPIs) play a crucial role in various biological processes, including signal transduction, transcriptional regulation, and metabolic pathways. Over the years, many methods have been developed to study PPIs, such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), pull-down assays, and surface plasmon resonance (SPR). However, each of these techniques has its own limitations, including false positives, a lack of specific binding partners, and restricted interaction zones. Fluorescence resonance energy transfer (FRET) has emerged as a powerful technique for investigating PPIs, offering several advantages over traditional methods. Recent advancements in fluorescence microscopy have further enhanced its application in PPI studies. In this review, we summarize recent developments in FRET-based approaches and their applications in PPIs research over the past five years, including conventional FRET, time-resolved FRET (TR-FRET), fluorescence lifetime imaging microscopy-FRET (FLIM-FRET), single-molecule FRET (smFRET), fluorescence cross-correlation spectroscopy FRET (FCCS-FRET), and provide guidance on selecting the most appropriate method for PPIs studies.