Skip to main content

Activity Measurements of Mitochondrial Enzymes in Native Gels

  • Protocol
Plant Mitochondria

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1305))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231

    Article  PubMed  Google Scholar 

  2. 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

    Article  Google Scholar 

  3. 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

    Article  CAS  PubMed  Google Scholar 

  4. 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

    Article  CAS  PubMed  Google Scholar 

  5. 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

    Article  PubMed  Google Scholar 

  6. 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. 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

    Article  PubMed  Google Scholar 

  9. Lojda Z, Gossrau R, Schiebler T (1979) Enzyme histochemistry: a laboratory manual. Springer, Berlin, pp 1–270

    Book  Google Scholar 

  10. Dubowitz V (1985) Muscle biopsy, a practical approach. Bailliere Tindall, London

    Google Scholar 

  11. 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

    Article  CAS  PubMed  Google Scholar 

  12. 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. 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

    PubMed Central  CAS  PubMed  Google Scholar 

  14. Schertl P, Cabassa C, Saadallah K et al (2014) Biochemical characterization of ProDH activity in Arabidopsis mitochondria. FEBS J 281:2794–2804

    Article  CAS  PubMed  Google Scholar 

  15. 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

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

We thank Dagmar Lewejohann for expert technical assistance.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hans-Peter Braun .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

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

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2639-8_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2638-1

  • Online ISBN: 978-1-4939-2639-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics