horseradish peroxidase (HRP), found in horseradish
, is used extensively in molecular biology
applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule. For example, an antibody
conjugated to HRP may be used to detect a small amount of a specific protein in a western blot
. Here, the antibody provides the specificity to locate the protein of interest and the HRP enzyme, in the presence of a substrate, produces a detectable signal. Horseradish peroxidase is also commonly used in techniques such as ELISA
Upon selected substrates, HRP produces derivatives of the labeled molecule allowing it to be detected and quantified by fluorescence or luminescence. Chemediate’s Kit uses a non-fluorescent substrate to quantify peroxidase in solution in presence of selected oxidizers Peroxidase catalyzes the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in a 1:1 stoichiometry with oxidizer (e.g. hydrogen peroxide) to produce a fluorescent product resorufin (Ex/Em = 560 nm/585 nm or absorbance at 570 nm). The assay can be performed in a convenient 96-well or 384-well microplate format and is amenable to either fluorescence or absorbance based detection of resorufin.