Stabilizing Protein and Protein-RNA Condensates in Low-Melting Agarose Hydrogels for Quantitative Biophysical Measurements.

Abstract

Biomolecular condensates formed by liquid-liquid phase separation are increasingly recognized as fundamental organizational units of the cell, with roles ranging from RNA processing to stress granule assembly. In vitro reconstitution of such condensates using purified proteins and RNA provides model systems, yet liquid droplets coarsen, sediment, and spread on surfaces over time, introducing significant variability into quantitative measurements over experimental timeframes. Embedding these condensates within a sparse, optically transparent, and chemically inert hydrogel-here prepared from low-melting agarose (LMA) at 0.3-1.0% w/v-stabilizes droplets in three dimensions, enabling the use of methodologies with longer measurement times or experimental setups with repeated measurements. This paper presents a detailed, step-by-step protocol for LMA stock preparation, droplet formation, and embedding, and provides examples of specific downstream biophysical readouts. We also show that specific droplet properties, such as molecular diffusion, are preserved after LMA embedding in the representative systems tested. Since the method is compatible with confocal, widefield, or super-resolution microscopy, fluorescence recovery after photobleaching, or nuclear magnetic resonance spectroscopy, among others, it can support quantitative characterization of biomolecular condensates relevant in health and disease.