Abstract
Zymography on peroxidase-entrapped gels enables the determination of the intrinsic specific activity (ISA) of H2O2-producing oxidases in the presence of interfering enzymes including catalase (decomposing hydrogen peroxide). To reveal the searched oxidase, the zymography gel is incubated first in the developing solution containing the appropriate substrate of the oxidase and an oxidizable chromogen such as o-phenylenediamine (o-PDA) peroxidase substrate. The zymographic gel, after image analysis, can be restained by Coomassie Blue for molecular weights, for the whole electrophoretic pattern and for the protein concentration, thus allowing for the determination of oxidase ISA.
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Abbreviations
- AO:
-
Amine oxidases
- APS:
-
Ammonium persulfate
- BSA:
-
Bovine serum albumin
- DAO:
-
Diamine oxidase
- GOx:
-
Glucose oxidase
- HRP:
-
Horseradish peroxidase
- ISA:
-
Intrinsic specific activity
- o-PDA:
-
ortho-Phenylenediamine
- PAA:
-
Polyacrylamide
- PAGE:
-
Polyacrylamide gel electrophoresis
- PBS:
-
Phosphate buffer solution
- SDS:
-
Sodium dodecyl sulfate
- TEMED:
-
N,N,N′,N′-Tetramethylethylenediamine
- Tris:
-
Trihydroxymethylaminomethane
References
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Acknowledgment
Financial support from NSERC (Natural Sciences and Engineering Research Council) of Canada (Discovery Program), from Fondation Courtois (Canada), from Regione Lazio of Italy (FILAS-RU-2014-1020), and from Joint Project Italia–Canada-Quebec (2017–2019), is gratefully acknowledged.
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Le, T.C., Mateescu, M.A., Ahmadifar, S., Marcocci, L., Pietrangeli, P. (2018). Zymographic Determination of Intrinsic Specific Activity of Oxidases in the Presence of Interfering Proteins. In: Kurien, B., Scofield, R. (eds) Protein Gel Detection and Imaging. Methods in Molecular Biology, vol 1853. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8745-0_24
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DOI: https://doi.org/10.1007/978-1-4939-8745-0_24
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