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Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue

Nature Protocols volume 5, pages 51–66 (2010)Cite this article

Abstract

Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24–48 h to complete.

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Figure 1: Antioxidant enzyme schematic.
Figure 2: MnSOD and CuZnSOD activity gels.
Figure 3: Catalase and GPx activity gels.
Figure 4: Antioxidant immunofluorescent staining of cultured cells.
Figure 5: Immunohistochemistry for MnSOD.
Figure 6: Subcellular localization of MnSOD by immunogold.

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Acknowledgements

This work was supported by an NIH Grant CA137230 and a VA Merit Review Grant.

Author information

Author notes

  1. Christine J Weydert and Joseph J Cullen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Christine J Weydert

  2. Department of Radiation Oncology, Free Radical and Radiation Biology Program, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Joseph J Cullen

  3. Holden Comprehensive Cancer Center, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Christine J Weydert & Joseph J Cullen

  4. Department of Surgery, The University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Joseph J Cullen

  5. VA Medical Center, Iowa City, Iowa, USA

    Joseph J Cullen

Authors
  1. Christine J Weydert
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Contributions

C.J.W. and J.J.C. discussed and commented on the paper at all stages.

Corresponding author

Correspondence to Joseph J Cullen.

Supplementary information

Supplementary Method

Method for fixing tissues and cultured cells for immunogold immunohistochemistry. (DOC 41 kb)

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Weydert, C., Cullen, J. Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 5, 51–66 (2010). https://doi.org/10.1038/nprot.2009.197

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