Denaturing SUMO Immunoprecipitation From Mitotic Cells.

Small ubiquitin-related modifiers (SUMOs) are covalently conjugated onto the proteome and serve as signaling molecules in many aspects of eukaryotic cell biology, from S. cerevisiae and C. elegans to H. sapiens. The conjugatable SUMO variants, SUMO1 and the almost identical SUMO2 and SUMO3 (designated SUMO2/3), are processed by an E1(SAE1:SAE2)-E2(UBC9)-E3 enzyme cascade to produce SUMO-modified proteins. The prerogative of the SUMO biology field is to identify and study the specific proteins undergoing SUMOylation, which grants us insights into the biological pathway of interest. This protocol was developed using the human osteosarcoma cell line U2OS to enable the investigation of SUMO conjugates in mitosis, the cell division phase of the cell cycle. We enrich the cell population for mitotic cells, which are isolated and subjected to stringent lysis conditions involving a high concentration of SDS and DTT in RIPA buffer, to promote complete protein denaturation. The lysates in high SDS RIPA buffer are diluted to reduce the overall SDS concentration and undergo conventional immunoprecipitation using SUMO1- or SUMO2/3-specific antibodies bound to protein A/G agarose beads. The samples are then compatible with downstream readouts such as western blots and mass spectrometry. This protocol detects endogenous SUMOylated proteins and avoids exogenous SUMO overexpression, which can alter SUMO conjugate formation. Furthermore, this denaturing protocol ensures only SUMOylated proteins are immunoprecipitated, and not their interactors. Key features • Purifies endogenous SUMO-modified proteins by building on Becker et al. [1]. • Enriches and isolates cells in mitosis using nocodazole and mitotic shake-off. • 1% SDS RIPA lysis promotes robust denaturation ahead of SUMO-specific immunoprecipitation. • Compatible with downstream readouts such as western blots and mass spectrometry.