ORAC assay measures antioxidant capacity

The Oxygen Radical Absorbance Capacity (ORAC) assay is a method that measures the antioxidant capacity of a substance. The ORAC assay measures a fluorescent signal from a probe that is quenched in the presence of Reactive Oxygen Species (ROS). Addition of an antioxidant absorbs the generated ROS, allowing the fluorescent signal to persist. Trolox® (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) is a vitamin E analogue and a known antioxidant. It is used as a standard by which all unknown antioxidants are compared. Modifications of the ORAC assay include the use of fluorescein as the fluorescent probe (ORACFL), the separation of hydrophilic and lipophilic antioxidants to obtain total antioxidant capacity and an adaptation to a high-throughput platform.

 

The ORAC assay is unique in that its ROS generator, AAPH (2,2’-azobis(2-methylpropionamidine) dihydrochloride), produces a peroxyl free radical upon thermal decomposition. This free radical is commonly found in the body, making this reaction biological relevant. Furthermore, AAPH is reactive with water and lipid soluble substances, so it can measure total antioxidant potential.

Application Notes

 

Examples of ORAC assays on BMG LABTECH microplate readers:

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