Protocol for combining immunological procedures with cell cycle stage synchronization to detect cell-cycle-specific...

Here, we present a protocol combining co-immunoprecipitation (Co-IP) and immunofluorescence approaches with cell cycle stage synchronization to detect cell-cycle-specific complexes. We describe steps to synchronize cells at specific cell cycle stages using drugs. We then detail the preparation of cell extracts from synchronized cells and fractionation of the protein complexes with density centrifugation, followed by Co-IP with specific antibodies. Protein-protein interactions are confirmed by localization using immunofluorescence imaging. This protocol is helpful for visualizing the dynamics of protein complex assembly. For complete details on the use and execution of this protocol, please refer to Habu and Kim (2021).¹.