Ponceau S as a Targeted Modulator for Protein Liquid–Liquid Phase Separation

Liquid-liquid phase separation (LLPS) in proteins is essential for cellular organization and biomolecular condensation. However, current methods to induce phase separation often lack precise spatiotemporal control. This study introduces Ponceau S as a selective modulator and monitors phase separation in bovine serum albumin and lysozyme. We demonstrate that Ponceau S effectively promotes the protein complex into liquid droplets by binding to hydrophobic regions and driving intermolecular interactions. Notably, Ponceau S fluorescence increases within protein-rich phases, reflecting the restricted molecular motion in these environments. Furthermore, the phase separation induced by Ponceau S is finely tunable through salt and 1,6-hexanediol adjustments, which influence droplet fusion and dissolution dynamics. This work highlights the potential of small molecules like Ponceau S to precisely regulate and monitor protein phase separation, providing a new dimension of control for applications in biomolecular engineering, drug delivery, and synthetic biology.