Ubiquitin reaction buffer

This protocol was adapted from a previous report.^{22 (link)} In brief, HA-c-Maf, HERC4, and Flag-USP5 plasmids were transfected into HEK293T cells, respectively. Forty-eight hours later, cells were treated with MG132 for 2 h, followed by cell lysate preparation. To enrich and purify c-Maf, HERC4, and USP5 proteins, individual cell lysates were subjected to IP with HA- (for c-Maf) or Flag- (for HERC4 and USP5) antibody-conjugating agarose beads, respectively, at 4 °C for 12 h. After that, the beads were washed four times with an IP lysis buffer, twice with 1 × ubiquitin reaction buffer (Boston Biochem, Boston, MA, USA) and then resuspended in 20 μl of 1 × ubiquitin reaction buffer containing 200 ng of recombinant E1, 250 ng of recombinant UbcH5c, 10 μg of ubiquitin, 0.5 mM ATP, and 1 × Energy Restoration System (Boston Biochem). The reaction was carried out at 30 °C for 2 h and then terminated by boiling in 2 × SDS loading buffer. ubiquitinated products were resolved by SDS-PAGE and detected by IB analysis.

This assay was adapted from a previous report [16 (link)]. Briefly, HA-c-Maf and Flag-UBE2O plasmids were transfected into HEK293T cells, respectively. Forty-eight hours later, cells were treated with MG132 for 2 h, followed by cell lysate preparation. To enrich and purify c-Maf and UBE2O proteins, individual cell lysates were subjected to immunoprecipitation with HA- (for c-Maf) or Flag- (for UBE2O) antibody-conjugating agarose beads, respectively, at 4 °C for overnight. After that, the beads were washed four times with an immunoprecipitation lysis buffer, twice with 1× ubiquitin reaction buffer (Boston Biochem, Boston, MA) and then re-suspended in 20 μl of 1× ubiquitin reaction buffer containing 200 ng of recombinant E1, 250 ng of recombinant UbcH5c, 10 μg of ubiquitin, 0.5 mM ATP, and 1× Energy Restoration System (Boston Biochem, Boston, MA). The reaction was carried out at 30 °C for 2 h and then terminated by boiling in the 2× SDS loading buffer. ubiquitinated products were resolved by SDS-PAGE and detected by immunoblotting analysis.