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Oxidative stress assays and oxidative stress markers

Related

  • Metabolism assays
    • Assays for mitochondrial research
      • Metabolism research resources
        • Oxidative phosphorylation pathway

          ​

          Learn about markers including ROS, MDA, GSH:GSSG, and more. Review assays for use with tissues, cells, and biofluids.

          Contents

          • Reactive oxygen species (ROS)
          • DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration
          • Antioxidants

          Reactive oxygen species (ROS)

          Reactive oxygen species (ROS) are reactive chemical species containing oxygen. They include peroxides, superoxide, hydroxyl radicals, singlet oxygen, and alpha-oxygen.

          Due to their transient nature, they are easily measured in live cells using fluorescent dye-based assays, such as with DCFDA. They are harder to measure in tissue and biofluid samples.


          Assay

          Readout

          Assay kits

          DCFDA – cellular reactive oxygen species

          Flow cytometry, plate reader

          ab113851

          Cellular superoxides

          Cellular ROS/Superoxide

          Microscope, flow cytometry

          ab139477

          ab139476

          Cellular reactive oxygen species

          Plate reader

          ab186027, ab186028, ab186029

          Cellular ROS/RNS

          Microscope

          ab139473

          Mitochondrial hydroxyl radical

          Mitochondrial superoxide

          Hydrogen peroxide

          Microscope, plate reader

          ab219931

          ab219943

          ab138874, ab138886, ab102500

          DCFDA web v1

          "We are currently using the product [ab113851] to measure microglial activation after 24 hours in response to activating stimuli. The product has been giving us very consistent results and is very easy to use.” 
          - Dr Neal Bennett


          DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration

          Oxidative stress can be measured indirectly by measuring the levels of DNA/RNA damage, lipid peroxidation, and protein oxidation/nitration, rather than a direct measurement of reactive oxygen species. These oxidative stress markers are more enduring than reactive oxygen species.

          DNA/RNA damage

          There are several types of DNA/RNA damage that can be measured as oxidative stress markers. 8-hydroxydeoxyguanosine (8-OHdG) is probably the most commonly used DNA damage marker for oxidative stress. Comet assays, assays for apurinic/apyrimidinic sites, and assays for aldehyde-induced damage can be used as less direct measures of DNA damage which is potentially related to oxidative stress.

          Lipid peroxidation

          Malondialdehyde (MDA) is the most commonly used lipid marker of oxidative stress. It is formed via peroxidation of polyunsaturated fatty acids and is typically quantified using the TBARS assay. The TBARS assay is not entirely specific for MDA, as other aldehydes also generate a signal with the assay, however, the TBARS assay is generally more convenient than using HPLC to measure MDA. Competitive ELISA assays for MDA are also available.

          Other lipid peroxidation markers include 4-HNA, 8-isoprostane, lipid hydroperoxides, and oxidized LDL.

          Protein oxidation / nitration

          Oxidative damage to proteins can take the form of protein carbonylation and protein nitration (3-nitrotyrosines). Reactive oxygen species can also cause the formation of advanced glycation end products (AGE) and advanced oxidation protein Products (AOPP). All of these markers can be measured by standard assays.


          Assay

          Readout

          Assay kits

          8-OHdG

          Plate reader

          ab201734

          Lipid hydroperoxide (LPO)

          Lipid peroxidation (MDA)

          Protein carbonyl content

          DNA damage –  apurinic/apyrimidinic  sites

          Plate reader

          ab133085

          ab118970

          ab126287

          ab211154

          Oxidized proteins

          Western blot

          ab178020











          Antioxidants

          Antioxidant enzymes and other redox molecules counteract the ROS that cause oxidative damage. There are three classes of antioxidants used as oxidative stress markers: small molecules, enzymes, and proteins (such as albumin).

          A number of assays exist to measure the total antioxidant capacity of a sample, including small molecule and protein antioxidant based capacity. One of the most common total antioxidant capacity assays is the Trolox equivalent antioxidant capacity assay (TEAC). The oxygen radical antioxidant capacity (ORAC) assay is another common oxidative stress assay that measures antioxidant capacity by measuring the ability of antioxidants to reduce the quenching of a fluorescent dye by ROS.

          Antioxidant activity can also be measured at the level of specific analytes. For instance by looking at the relative levels of GSH and GSSG. Glutathione (GSH) is considered the most abundant molecule among endogenous antioxidants, forming GSSG in its oxidized form. It is recycled by glutathione reductase.

          Otherwise, the levels of activity of antioxidant enzymes, such as GST and Superoxide dismutase can be measured in relation to the levels of oxidative stress.

          Assay

          Readout

          Assay kits

          Total antioxidant capacity: copper-based

          Total antioxidant capacity - FRAP assay (Ferric Reducing Antioxidant Power Assay

          Plate reader

          ab65329


          ab234626

          Ascorbic acid

          NAD/NADH

          NADP/NADPH

          GSH/GSSG ratio

          Thiol

          Plate reader

          ab65656

          ab65348, ab176723

          ab65349, ab176724

          ab138881, ab205811

          ab112158, ab219272

          Intracellular glutathione (GSH)

          Flow cytometry, plate reader

          ab112132

          GST

          Superoxide dismutase

          Glutathione reductase

          Xanthine oxidase

          Glutathione peroxidase

          Aconitase

          Catalase

          Thioredoxin reductase

          NQO1

          Peroxidase

          Plate reader

          ab65325, ab65326

          ab65354

          ab83461

          ab102522

          ab102530

          ab109712

          ab118184, ab83464

          ab190804

          ab184867

          ab155895

          Oxidative stress defense cocktail (catalase, SOD1, TRX, smActin)

          Western blot

          ab179843



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