Construction of Complete Phase Diagrams for Protein Liquid–Liquid Phase Separation by <i>In Situ</i> Raman Microspectroscopy

Liquid-liquid phase separation of biomolecules can be induced <i>in vitro</i> by selecting specific concentrations and environmental conditions. Phase diagrams relating the temperature, pressure, etc. to the concentrations of proteins and co-solutes aid in identifying the specific phase separation conditions for target proteins. However, it remains challenging to determining complete phase diagrams for protein liquid-liquid phase separation. Here, we combine optical microscopy and <i>in situ</i> Raman spectroscopy to quantify the temperature-dependent concentrations of the light and dense phases of human γD-crystallin and construct a complete phase diagram. The method can be extended to systems in the presence of co-solutes, which enables the quantitative description of the partitioning of a co-solute in the light and dense phase of proteins.