Cells use various strategies to organize biochemical processes in both space and time. In addition to membrane-bound organelles, proteins and nucleic acids can undergo liquid-liquid phase separation (LLPS) to form dynamic, membraneless compartments known as biomolecular condensates. These micron-sized, liquid-like structures are particularly enriched in RNA-binding proteins that contain RNA-binding domains (RBDs) and intrinsically disordered regions (IDRs) that are prone to LLPS. Such condensates play crucial roles in RNA metabolism. They are also linked to neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, where stress conditions may lead to their abnormal maturation into solid aggregates.

Despite the increasing interest in this area, much remains unknown about the fundamental biophysical properties of multi-component protein-RNA condensates, especially in complex biological environments. The challenge is further complicated by the limitations of any single technique to fully capture the behavior and dynamics of these systems. Therefore, an integrative approach combining various methods and theoretical frameworks is essential for comprehensively characterizing these condensates.

This Methods Collection aims to present a diverse range of biophysical and imaging techniques for probing multi-component biomolecular condensates, both in vitro and in vivo. These standardized protocols are designed to serve as practical tools for investigating the formation, composition, material properties, and dynamics of these condensates, providing cross-disciplinary insights into the role of LLPS in health and disease.