Capillary Flow Experiments (Capflex) for Thermodynamic and Kinetic Characterization of Protein LLPS at High Throughput

Liquid-liquid phase separation (LLPS) of proteins is a field of mounting importance to life sciences. We present a new method, Capflex, that can easily be automated, allowing rapid and accurate quantification of key parameters for LLPS. Dilute phase concentration, relative droplet size distributions and the kinetics of droplet formation are quantified. Uniquely, the binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We applied Capflex to characterize the LLPS of Ddx4n1 and found that PEG3000 and Ca²⁺ promotes LLPS while ssDNA is detrimental. Furthermore, we characterized the membraneless organelle model system RP₃ and provide the first experimentally recorded affinity of RP₃ for DNA. We believe the high information content and high throughput of Capflex makes it a valuable tool for characterizing biomolecular LLPS.