Protocol for multi-component reconstitution of stress granules in vitro

To date, stress granule studies mainly focus on cell models. The diversity of molecules in stress granules makes it challenging to uncover the function of each molecule in stress granule regulation. Here, we provide a protocol to reconstitute stress granules with multi-components in vitro. We describe steps for strain amplification, protein purification, and liquid-liquid phase separation (LLPS). The multi-component reconstitution system constructed in this protocol also provides a technique for other condensate reconstitution studies in vitro. For complete details on the use and execution of this protocol, please refer to Yao et al.¹.