Abstract
In-gel activity assays are useful tools to identify and characterize enzymes within gels. Prerequisite are electrophoretic protein separations that are carried out under conditions compatible with enzyme activity. While blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for activity assays of the five enzyme complexes of the oxidative phosphorylation system, the blue background of this electrophoretic system is not compatible with activity assays for some other mitochondrial enzymes. As an alternative system, clear native (CN)-PAGE can be used for visualizing activities of mitochondrial enzymes. Here, we describe enzyme activity assays for mitochondrial enzymes in BN and CN gels.
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References
Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231
de St F, Groth S, Webster RG, Datyner A (1963) Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochim Biophys Acta 71:377–391
Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064
Jung C, Higgins CM, Xu Z (2000) Measuring the quantity and activity of mitochondrial electron transport chain complexes in tissues of central nervous system using blue native polyacrylamide gel electrophoresis. Anal Biochem 286:214–223
van Coster R, Smet J, George E et al (2001) Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr Res 50:658–665
Sabar M, Gagliardi D, Balk J, Leaver CJ (2003) ORFB is a subunit of F1F(O)-ATP synthase: insight into the basis of cytoplasmic male sterility in sunflower. EMBO Rep 4:381–386
Schertl P, Sunderhaus S, Klodmann J et al (2012) L-galactono-1,4-lactone dehydrogenase (GLDH) forms part of three subcomplexes of mitochondrial complex I in Arabidopsis thaliana. J Biol Chem 287:14412–14419
Schägger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217:220–230
Lojda Z, Gossrau R, Schiebler T (1979) Enzyme histochemistry: a laboratory manual. Springer, Berlin, pp 1–270
Dubowitz V (1985) Muscle biopsy, a practical approach. Bailliere Tindall, London
Smet J, de Paepe B, Seneca S et al (2011) Complex III staining in blue native polyacrylamide gels. J Inherit Metab Dis 34:741–747
Seligman AM, Karnovsky MJ, Wasserkrug HL et al (1968) Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol 38:1–14
Cox GB, Downie JA, Fayle DR et al (1978) Inhibition, by a protease inhibitor, of the solubilization of the F1-portion of the Mg2+-stimulated adenosine triphosphatase of Escherichia coli. J Bacteriol 133:287–292
Schertl P, Cabassa C, Saadallah K et al (2014) Biochemical characterization of ProDH activity in Arabidopsis mitochondria. FEBS J 281:2794–2804
Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225
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We thank Dagmar Lewejohann for expert technical assistance.
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Schertl, P., Braun, HP. (2015). Activity Measurements of Mitochondrial Enzymes in Native Gels. In: Whelan, J., Murcha, M. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 1305. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2639-8_9
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DOI: https://doi.org/10.1007/978-1-4939-2639-8_9
Publisher Name: Humana Press, New York, NY
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