SUMO Antibody Validation.

Tools to detect endogenous SUMOylation are critical for capturing and analyzing SUMOylation dynamics. Proteome-wide analysis of SUMOylation has provided invaluable insight into our understanding of global patterns of SUMOylation under various developmental and stressor conditions. Experimental validation of SUMOylation is still essential for a detailed mechanistic understanding of SUMOylation of individual proteins. In most cases, antibodies raised to SUMO are the tools of choice. Several high-quality monoclonals and their hybridomas (8A2 to SUMO2/3, 21C7 and 76-86 for SUMO1) have been made widely available to the research community (Becker et al., Nat Struct Mol Biol 20:525-531, 2013). These tools are helpful in identifying individual substrates and as affinity reagents for global proteomic SUMOylome profiling. In addition to these tools, many SUMO1-4 antibodies are commercially available, including almost 100 monoclonal antibodies (MAbs). In testing 24 SUMO1-4 MAbs across multiple detection formats (Garvin et al., Sci Rep:12, 2022), we uncovered several poorly specific published MAbs, including SUMO4 MAbs, that robustly cross-react with SUMO2/3 and vice versa. Inconsistencies between different SUMO MAbs were prevalent in their ability to detect monomeric, conjugated, and polymeric forms of SUMO. The ability to detect changes in SUMOylation across stress conditions also varied widely between SUMO MAbs. As each SUMO MAb tested had strengths and weaknesses, researchers must be aware of these caveats when selecting antibodies for their research needs. This chapter provides protocols for validating SUMO antibodies. As many vendors provide little experimental validation for their antibodies, these validations are critical to prevent wasting precious time and resources for researchers. The proper validation of antibodies is also increasingly a prerequisite for research funding agencies, publishing, and promoting reproducibility in research.