4.2.2. Production of Di-Ubiquitin Chains

Di-ubiquitin chains (Ub2) were obtained from Ub variants through enzymatic reactions, with the strategy described in [46,47]. The reaction bu ffer contained 50 mM Tris HCl pH 8.0, 5 mM MgCl2, 10 mM ATP, 3 mM TCEP, 0.02% NaN3 and protease inhibitors. The ubiquitin variants K48R+D77 and K48R+UbSH were used as building blocks for the reconstitution of Ub2(48) and Ub2(48)-SH, respectively. For the assembly of these K48-linked chains, we used 1 μM E1 and 20 μM E2-25K. The ubiquitin variants K63R+D77 and K63R+UbSH were used to assemble Ub2(63) and Ub2(63)-SH, respectively. For these K63-linked chains, we used 1 μM E1, 25 μM Ubc13 and 25 μM Mms2. After reaction, we incubated the solutions with 0.2% perchloric acid to obtain precipitation of most of the enzymes. Then, SP cation-exchange and superdex-75 gel filtration were performed to obtain the pure di-ubiquitin chains.
