Q: How does this assay work?
A: Our Total Antioxidant Capacity Assay is a colorimetric assay that quantifies antioxidant activity via a SET mechanism and is based on copper reduction. Copper (II)-bathocuproine is used as the chromogenic reagent and the assay limit of detection is 0.0039mM uric acid.
Q: Can you please explain the difference between the ORAC, HORAC, and TAC assays and why I would select one over the other?
All three assays measure antioxidant capacity but are slightly different. The ORAC and HORAC assays are both fluorometric assays that use HAT chemistry to detect oxidation of a fluorescent probe. The main difference between the two assays is the free radicals that are generated that oxidize the probe. The ORAC assay uses a free radical initiator to produce peroxyl radicals, while the HORAC assay uses a hydroxyl radical initiator and fenton reagent to produce hydroxyl radicals (HORAC = hydroxyl radical antioxidant capacity, ORAC = oxygen radical antioxidant capacity). The TAC assay is a colorimetric assay that uses a different chemistry than the ORAC and HORAC, specifically a SET mechanism that is based on copper reduction by antioxidants. The ORAC and HORAC assays are more sensitive than the TAC assay, but they are more technically demanding in both assay protocol and data analysis.
The choice between assays is dependent upon the chemistry that is most applicable to the research being performed (ex: SET vs. HAT), or the free radical generated (ex: hydroxyl vs. peroxyl). Researchers tend to prefer ORAC over HORAC because peroxyl radicals appear to be more relevant than hydroxyl radicals.
Q: Can this kit be used with samples from any species?
A: Yes, our Total Antioxidant Capacity Assay is not species specific and should work with any biological protein sample because it measures activity rather than protein structure.
Q: Is it possible to test samples that have been stored frozen?
A: Two months at -80ºC generally the longest most antioxidant samples should be stored, but we recommend testing fresh samples wherever possible for the best results.
Q: Does hydrophilic/lipophilic refer to the samples or the antioxidants as discussed in the product manual?
A: Sample preparation is based on the solubility properties of the sample. With samples that contain many different antioxidants, such as plants or food, we recommend preparing both fractions because not all antioxidants will be soluble in each fraction.
Q: Are serum/plasma/tissue lysate samples hydrophilic or lipophilic? Do you have a detailed protocol for preparing samples?
A: There are different antioxidants in each fraction and we can’t recommend preparing one over the other. We suggest preparing both fractions, however if only one fraction can be prepared, most researchers test the hydrophilic fraction.
Q: What does 1:2, w/v mean under preparation of food samples?
A: Food samples used with our Total Antioxidant Capacity Assay should be prepared as 1 g sample : 2 mL deionized water.
Q: What is the protein concentration I should use with cell lysates?
A: We are unable recommend a protein concentration for the Total Antioxidant Capacity assay because the antioxidant capacity will vary between samples and will be influenced by cell type, sample preparation, and storage conditions, making it difficult for us to know what will work for each sample. Our recommendation is to start with the most concentrated sample possible and perform a sample titration prior to performing the experiment to determine any necessary dilutions so that the samples fall within the standard curve.
Q: Can citrate plasma samples be used with this assay?
A: Citrate plasma samples are compatible with the Total Antioxidant Capacity assay. EDTA should be avoided since this will interfere with the assay.
Q: Can this kit measure the total antioxidant capacity of cell culture supernatant?
A: Unfortunately our Total Antioxidant Capacity Assay is not compatible with cell culture supernatants. This assay is based on the reduction of copper and the iron present in the media will interfere with this reaction. You may want to consider using our ORAC Assay which uses a different chemistry and is more sensitive than our TAC Assay. The ORAC Assay is compatible with cell culture supernatants, but you will want to use a control of the media to determine the level of background interference.
Q: How are the results calculated?
A: The net ODs are first calculated by subtracting initial ODs from final ODs. The net ODs of the standards are then plotted to generate a standard curve (OD vs concentration). A linear trendline is generated and the equation of the line (y=mx+b) can be used to calculate the mM uric acid equivalent (UAE) for each sample by solving for y, which is (OD value-b)/m.