Abstract
We have chemically synthesised a number of ubiquitin extension proteins, with carboxyl-terminal single amino acid residue extensions, to use as substrates to assess the catalytic capacities of deubiquitinating enzymes (DUBs). Here we describe a modified acrylamide gel electrophoresis system which allows separation of peptide- or isopeptide-linked ubiquitin-lysine from ubiquitin (77 and 76 residue proteins respectively) in only 2 h. Western blotting, using antibodies against ubiquitin, allows both substrate (i.e. ubiquitin-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.
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