Electrophoretic separation of ubiquitin and single amino acid residue ubiquitin extensions using a commercial modified acrylamide gel electrophoresissystem: An assay to determine catalytic capacities of deubiquitinating enzymes

We have chemically synthesised a number of ubiquitin extension proteins, wi th carboxyl-terminal single amino acid residue extensions, to use as substr ates to assess the catalytic capacities of deubiquitinating enzymes (DUBs). Here we describe a modified acrylamide gel electrophoresis system which al lows separation of peptide- or isopeptide-linked ubiquitin-lysine from ubiq uitin (77 and 76 residue proteins respectively) in only 2 h. Western blotti ng, using antibodies against ubiquitin, allows both substrate (i.e. ubiquit in-lysine) and product (i.e. ubiquitin) of DUB-catalyzed cleavage reactions to be detected. Catalytic capacities of DUBs may be indicative of in vivo functions of these proteases.