Measuring Partition Coefficients of In Vitro Biomolecular Condensates Using Fluorescence Correlation Spectroscopy

Liquid-liquid phase separation is a widespread organizing principle of live cells, including, for example, the spatiotemporal organization of bacterial chromatin. The biophysics of phase-separating systems is often studied in vitro to avoid the complexity of the live-cell environment and facilitate application of advanced biophysical methods. One attractive method for measuring, e.g., partition coefficients, in such systems is fluorescence correlation spectroscopy (FCS). FCS circumvents some of the limitations of the widespread confocal laser scanning microscopy image-based measurements. Here, we describe how to perform partition coefficient measurements in biological phase-separating systems. Our protocol details typical workflows for the preparation of in vitro reconstituted condensates, FCS data acquisition, and subsequent data analysis, including corrections of some common artifacts. Our recommendations should help avoid many pitfalls of partition coefficient determination in these challenging systems.