On 29 June 2023, the European Union’s (EU) Markets in Crypto Assets Regulation (MiCA) entered into force. It is being implemented in stages depending on the provisions, between June and December 2024. The regulation, which provides clearer rules for crypto-asset service providers and token i...
Briefly, NPL4 and UFD1-His6 proteins were expressed individually (ratio 2:1) overnight at 18 °C in chemically competent E. coli BL21(DE3) (Thermo Fisher Scientific) by addition of 1 mM IPTG (AppliChem). Bacterial pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0; 0.5...
and 10 mM MgCl2at 37 °C overnight. To purify ubiquitinated Eos3.2 from free ubiquitin chains, the reaction mixture was incubated with Ni-NTA resin, eluted with 300 mM imidazole, and run over a Superdex 200 size-exclusion column equilibrated in the SEC Buffer. Fractions with long ubiq...