Reversible attachment of SUMO (small ubiquitin related modifi er) regulates a large number of proteins and plays an important role in processes such as transcriptional regulation, nucleo-cytoplasmic transport, genome integrity, and cell cycle progression. The steady state level of most sumoylated proteins is very low, presumably caused by strictly regulated modifi cation and/or rapid cycles of modifi cation and de-modifi cation. This often causes a detection problem of sumoylation in vivo. One approach to overcome this obstacle is described here and involves enrich ment of sumoylated proteins under denaturing conditions. After sumoylation is veri fied, addressing its functional consequences is the logical next step. This will benefit signifi cantly from the availability of large quantities of modifi ed protein. A protocol for effi cient in vitro sumoylation of target proteins is described here. It makes use of an E3 ligase fragment that functions without target discrimination.