Concentration Quantification of the Low-Complexity Domain of Fused in Sarcoma inside a Single Droplet and Effects of Solution Parameters

Liquid–liquid phase separation (LLPS) is an important phenomenon in biology, and it is desirable to develop quantitative methods to analyze protein droplets generated by LLPS. This study quantified the change in protein concentration in a droplet in label-free and single-droplet conditions using Raman imaging and the Raman band of water as an intensity standard. Small changes in the protein concentration with variations in pH and salt concentration were observed, and it was shown that the concentration in the droplet decreases as the conditions become less favorable for droplet formation. The effect of exposure to 1,6-hexanediol was also examined, and this additive was found to decrease the protein concentration in the droplet. A model can be proposed in which the addition of 1,6-hexanediol reduces the protein concentration in the droplet, and the droplet disappears when the concentration falls below a certain threshold value.