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

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

Nature protocols (2010-01-09)
Christine J Weydert, Joseph J Cullen
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium hydroxide, reagent grade, ≥98%, pellets (anhydrous)
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β-Nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, ≥95% (HPLC)
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Nitrotetrazolium Blue chloride, ≥90.0% (HPLC)
Sigma-Aldrich
N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ≥99.0%
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Bromophenol Blue, titration: suitable