title: Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer.
abstract: Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins.
authors.affiliation: Centre for Cell Biology and Cutaneous Research, Blizard Institute, Queen Mary University of London, London, Great Britain.
authors.forename: Sreekanth
authors.initials: S
authors.lastname: Vootukuri Reddy
authors.affiliation: Centre for Cell Biology and Cutaneous Research, Blizard Institute, Queen Mary University of London, London, Great Britain.
authors.forename: Mike P
authors.initials: MP
authors.lastname: Philpott
authors.affiliation: Centre for Cell Biology and Cutaneous Research, Blizard Institute, Queen Mary University of London, London, Great Britain. g.trigiante@qmul.ac.uk.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Trigiante
mesh_terms: D001586:Benzoylarginine Nitroanilide / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D057056:Cysteine Proteases / Q000737:chemistry* / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D008956:Models, Chemical; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism
chemical_list: D002021:Buffers; D017382:Reactive Oxygen Species; D001586:Benzoylarginine Nitroanilide; D057056:Cysteine Proteases; D003545:Cysteine
keywords: Cysteine proteases; L-BApNA; Zymography