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.
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.
Layfield
R
R
School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, United Kingdom. robertlayfield@nottingham.ac.uk
Hayes
C
C
Wang
P
P
Urquhart
K
K
Ramage
R
R
Mayer
R J
RJ
Landon
M
M
D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism*; D014452:Ubiquitins / Q000302:isolation & purification*
D014452:Ubiquitins; D010450:Endopeptidases