Protocol to alter a protein's phase separation capacity to control cell fate transitions

Abstract

Phase separation of proteins regulates transcription. Here, we present a protocol to manipulate phase separation capacity of a protein. We use this protocol to disrupt phase separation by mutating residues at intrinsically disordered regions (IDRs). Further, we rescue the disabled phase separation by fusing an IDR known to drive phase separation. Phase separation promotes cell fate transitions, whereas disruption of phase attenuates the transitions. The major challenge is how to effectively predict mutation residues. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Keywords: Cell Biology; Cell-based Assays; Microscopy; Molecular Biology; Molecular/Chemical Probes; Protein expression and purification; Stem Cells.