OxiSelect™ Hydrogen Peroxide / Peroxidase Assay Kit (Fluorometric)User Manual
|Research Area:||Oxidative Stress|
|Assay Category:||Hydrogen Peroxide Assays|
|Sample Type:||Suitable for use with cell lysates, tissue homogenates, cell culture supernatants, plasma, serum, urine, or other biological fluids|
The Oxiselect Hydrogen Peroxide/Peroxidase Assay Kit is a simple HTS-compatible assay for measuring hydrogen peroxide concentrations or peroxidase activities in biological samples without any need for pretreatment. In the presence of hydrogen peroxide and horseradish peroxidase (HRP), non-fluorescent ADHP (10-Acetyl-3, 7-dihydroxyphenoxazine) is oxidized to the highly fluorescent Resorufin. The probe has less background and greater stability. The ADHP-based hydrogen peroxide detection is at least one order of magnitude more sensitive than the commonly used Xylenol Orange (FOX) colorimetric assay for hydrogen peroxide. The probe can be also used as an ultrasensitive assay for peroxidase activity when hydrogen peroxide is in excess. Due to the high extinction coefficient of Resorufin (58,000 ± 5000 cm-1M-1), the assay could be read fluorometrically as well as spectrophotometrically. The kit has a detection sensitivity limit of 50 nM (hydrogen peroxide) or 0.1 mU/ml (Peroxidase). Each kit provides sufficient reagents to perform up to 500 assays, including standard curve and unknown samples.
The Kit is a sensitive quantitative fluorometric assay for hydrogen peroxide or peroxidase. In the presence of HRP, ADHP reacts with hydrogen peroxide in a 1:1 stoichiometry to produce highly fluorescent Resorufin. The Resorufin product can be easily read by a fluorescence microplate reader with an excitation of 530-560 nm and an emission of 590 nm, or for absorbance at 560 nm. Fluorescence values are proportional to the hydrogen peroxide or peroxidase levels within the samples. The hydrogen peroxide or peroxidase content in unknown samples is determined by comparison with its respective standard curve.
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