title: Electrophoretic separation of ubiquitin and single amino acid residue ubiquitin extensions using a commercial modified acrylamide gel electrophoresis system: an assay to determine catalytic capacities of deubiquitinating enzymes.
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.
authors.affiliation: School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, United Kingdom. robertlayfield@nottingham.ac.uk
authors.forename: R
authors.initials: R
authors.lastname: Layfield
authors.forename: C
authors.initials: C
authors.lastname: Hayes
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: K
authors.initials: K
authors.lastname: Urquhart
authors.forename: R
authors.initials: R
authors.lastname: Ramage
authors.forename: R J
authors.initials: RJ
authors.lastname: Mayer
authors.forename: M
authors.initials: M
authors.lastname: Landon
mesh_terms: D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism*; D014452:Ubiquitins / Q000302:isolation & purification*
chemical_list: D014452:Ubiquitins; D010450:Endopeptidases