Protocol for in vitro phase separation of N terminus of CIDEC proteins

Cell death-inducing DFF45-like effector C (CIDEC) condensation through N-terminal phase separation is a prerequisite for CIDEC-mediated lipid droplet (LD) fusion and lipid storage promotion. Here, we present a protocol for in vitro phase separation of the N terminus of CIDEC (CIDEC-N) proteins. We describe steps for plasmid construction, protein expression, and purification. We then detail procedures for concentration detection and in vitro phase separation. This protocol provides a framework to investigate CIDEC-N phase separation properties, laying the foundation for further exploration of its role in lipid metabolism and obesity. For complete details on the use and execution of this protocol, please refer to Lyu et al.¹.