Crowder titrations enable the quantification of driving forces for macromolecular phase separation.

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. In addition, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation.