Protocol for investigating phase transition of prion-like proteins in vitro with concentration variation.

Protein concentration is an important factor regulating liquid-to-solid phase transition of proteins with prion-like domains (PLDs). Here, we present a protocol for investigating the effect of prion-like protein concentration on phase transition of fibrillarin (FBL) condensates in vitro using phase separation assays. We describe steps for construction of fluorescent-protein-tagged FBL plasmids, protein expression and purification in vitro, and detection of condensed states of FBL. This protocol has potential applications in phase separation assays for studying phase transitions in any prion-like protein. For complete details on the use and execution of this protocol, please refer to Sun et al.(1).