Q: I want to test my samples for oxidative stress, but there are so many oxidative stress markers. How do I know which assays to use?
A: Oxidative stress may be tested either directly, by measuring the presence of various reactive oxygen species, or indirectly by measuring the resulting damage on one or more biomolecules (DNA, RNA, proteins or lipids). The indirect method is more common because of the stability of most markers of oxidative damage and the relative instability of many ROS.
Many journal reviewers are requesting confirmation of oxidative stress via testing of multiple oxidative stress markers. This may be accomplished by testing multiple markers on one biomolecule (e.g. different markers of protein damage), or by testing one marker each on different molecules (e.g. protein, lipid and DNA).
Here are the best assays to begin your oxidative stress studies:
You may also use our Oxidative Stress Assay Selection Guide.
Q: Do Reactive Oxygen Species form outside of oxidative stress?
A: ROS are produced outside of oxidative stress as part of normal metabolism, and these assays do not differentiate between the source of ROS production. However, an increase in ROS/damage levels is expected when samples are subjected to stress. Most oxidative stress studies measure relative changes in ROS production rather than absolute quantitation.
Q: What is the advantage or disadvantage to measuring ROS directly compared to damage markers on proteins and DNA?
A: Although direct measurement of ROS is ideal, because of their transient nature ROS can be difficult to detect directly. Many researchers instead prefer to measure damage markers because they tend to be more stable and therefore more easily and reliably measured.
A: Nearly all of our OxiSelect™ assays are species-independent, which means they will work on samples from any species. This is because the assays are specifically measuring the damage marker, not the biomolecule itself. Specific proteins, for example, may vary across different species, but markers of protein damage such as protein carbonyl and 3-nitrotyrosine are always the same.
Q: What is the difference between a Competitive ELISA and other ELISA formats? Why do I need to measure and adjust the protein concentration of my samples for some of your oxidative stress ELISA kits and not for others such as Competitive ELISA kits?
A: We offer three different types of ELISA kits: sandwich ELISA, competitive ELISA, and indirect ELISA. With an indirect ELISA you coat your samples directly onto a protein binding plate, rather than on a plate precoated with an antibody as is done with a sandwich ELISA. Because the protein binding capacity of the plate is not linear, it is important to maintain the same protein concentration in all standards and samples to allow direct comparison between your unknowns and the standard curve. For our indirect ELISA kits all samples and standards must be diluted to 10 µg/mL total protein, which means a protein assay is required for all samples.
For competitive ELISA kits it is not necessary to measure the protein concentration of your sample, because your sample does not bind to the plate. Rather it competes with the target antigen on the plate for the attention (i.e. binding) of the primary antibody.
Q: How do I prevent my samples from becoming further oxizided during storage?
A: To prevent further oxidation during storage, 0.005% BHT can be added to samples. A 5% solution of butylated hydroxytoluene (available from Sigma-Aldrich) in methanol can be prepared as the stock solution (1000X).
Q: What is the most reliable assay to detect lipid peroxidation in vitro?
A: We offer several assays for the following lipid peroxidation markers: 4-HNE, 8-isoprostane, MDA, and oxidized LDL. We offer ELISA kits for HNE, isoprostane, MDA, and oxidized LDL markers. Our HNE Adduct ELISA and MDA ELISA are similar kits but the MDA adduct is not as stable and samples should be stored at -80ºC for no more than one month. The OxLDL ELISAs are specific for either CML, HNE, or MDA that is on LDL, while the MDA and HNE Adduct ELISAs are specific for those markers on any protein. The best assay will depend on the marker of interest and sample type. In general, we recommend the HNE Adduct Competitive ELISA kit (STA-838), which is one of our most popular kits and the marker is relatively stable.