Preparation and Characterization of Thiocarboxylated Urm1

Uba4‐mediated thiocarboxylation of Urm1 was performed as described (Termathe & Leidel, 2018 (link)). Briefly, 20 μM of Urm1 was mixed with 10 μM of Uba4 in the thiocarboxylation buffer (20 mM HEPES pH 8.5, 150 mM NaCl, and 2 mM MgCl₂), supplemented with 5 mM ATP, 5 mM TCEP and 180 μM of sodium thiosulfate (Na₂S₂O₃) was added to the reaction mix and incubated for 1 h at 30°C. For Urm1‐SH, samples were desalted using PD SpinTrapTM G‐25 (Cytiva) columns and either loaded on SDS–PAGE gels supplemented with 20 μM of APM to visualize the shift of the thiol group or buffer exchanged into 40 mM ammonium acetate and analyzed by ESI‐MS. Thiocarboxylation of Urm1 was scaled up and Urm1‐SH was purified by using Superdex 200 Increase 10/300 GL (Cytiva) on ÄKTA™ start system. The purified Urm1‐SH was snap frozen and stored at −80°C in storage buffer (20 mM Tris pH 7.5 and 200 mM NaCl) for further use.