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Abstract

Protein regulation by reversible attachment of SUMO (small ubiquitin-related modifier) plays an important role in several cellular processes such as transcriptional regulation, nucleo-cytoplasmic transport, cell-cycle progression, meiosis, and DNA repair. However, most sumoylated proteins are of marginal abundance at steady state levels, which is due to strict regulation and/or rapid turnover of modification and de-modification. Consequently, analysis of protein sumoylation in vivo is very challenging. Nonetheless, a novel method was established that allows detection of sumoylated proteins at endogenous levels from vertebrate cells and tissues. This approach involves the enrichment of sumoylated proteins by immunoprecipitation followed by peptide elution. After endogenous substrate sumoylation is verified, addressing its functional consequences is the next logical step. This requires SUMO site mapping that benefits from larger quantities of modified protein. Here, we shortly describe strategies to achieve efficient in vitro sumoylation of many substrates.