Cardiovascular and Oxidative Stress Assay Kit

HORAC Microplate Assay Kit

$385.00

The HORAC Microplate Assay Kit is for the quantitative determination of Hydroxyl Radical Antioxidant Capacity in biological fluids by a microplate assay. The HORAC Microplate Assay Kit is for research use only and not for use in diagnostic procedures.

SKU: HOR39-K01 Categories: , ,

HORAC Microplate Assay Kit

The HORAC Microplate Assay Kit is For Research Use Only

Size: 1×96 wells
Sensitivity: 0.1 ng/mL
Dynamic Range: 0.1 – 100 ng/mL
Incubation Time: 1.5 hour
Sample Type: Biological Fluids
Sample Size: 100 µl
Alternative Name: Hydroxyl Radical Antioxidant Capacity

Product manufactured in the USA

Assay Background

It is now well established that oxidative stress is a major risk factor for the development of several diseases including atherosclerosis, cardiovascular disease, and cancer. Oxidative stress is the condition in which there is an imbalance between the concentrations of reactive oxygen species (ROS) and physiological antioxidants, resulting in oxidative damage to many biomolecules within the cell. Products of ROS-mediated oxidation are widely used to monitor oxidative stress. However, it is also important to assess the antioxidant capacity of cells and biological fluids, as well as putative “functional foods” to assess their antioxidant capacity. Organisms possess multiple antioxidant systems to help regulate ROS and prevent oxidative stress. In vertebrates, these include enzymes that metabolize ROS, antioxidant proteins, and smaller molecules that are important antioxidants. These antioxidants include hydrophilic as well as lipid-soluble molecules that are localized throughout various tissues and cell types.

Classification Examples
Enzymes: superoxide dismutase, catalase, glutathione peroxidase
Large Molecule: albumin, ferritin, ceruloplasmin
Small Molecule: ascorbic acid, α-tocopherol, β-carotene, uric acid, bilirubin, glutathione, methionine

Given the multiplicity of antioxidant pathways, their centrality in the prevention of oxidative stress, and the influences of lifestyle and nutritional supplements on an individual’s antioxidant capacity, it is important to be able to quantitatively measure the total antioxidant capacity or antioxidant power in a biological specimen or in nutrients.

Related Products

Total Antioxidant Power Microplate Assay
GSH GSSG Microplate Assay
Creatinine Microplate Assay Kit

Additional Information

Assay Principle

The Hydroxyl Radical Antioxidant Capacity (HORAC) assay is based on the oxidation of fluorescein by hydroxyl radicals via a classic hydrogen atom transfer (HAT) mechanism. Free radicals are generated by hydrogen peroxide (H2O2). The hydroxyl radicals thus generated quench the fluorescence of fluorescein over time. The antioxidants block the hydroxyl radical-mediated oxidation of fluorescein until all of the antioxidant activity in the sample is exhausted, after which the H2O2 radicals react with and quench the fluorescence of fluorescein. The area under the fluorescence decay curve (AUC) is used to quantify the total hydroxyl radical antioxidant activity in a sample and is compared to a standard curve obtained using various concentrations of gallic acid. Unlike other antioxidant activity assays, the fluorescent Hydroxyl Radical Antioxidant Capacity (HORAC) assay provides a direct measurement of antioxidant capacity against hydrophilic chain-breaking hydroxyl radicals.

  1. Place 20 µL of Sample or Standard into the appropriate wells of the provided microplate. See the Suggested Plate Layout on the next page for a potential organization of the assay.
  2. Place 140 µL of the Fluorescein Working Solution into each of the wells.
  3. Incubate the plate in the fluorescent plate reader for 30 minutes at 37°C. It is suggested that a sample reading be taken at the beginning and end of this incubation at an excitation wavelength of 488 nm and an emission wavelength of 515 nm.
  4. Place 20 µL of the Hydroxyl Radical into each well using either a multichannel pipette or built-in dispensing unit of the fluorescent plate reader.
  5. >Place 20 µL of the Fenton Reagent into each well using either a multichannel pipette or built-in dispensing unit of the fluorescent plate reader.
  6. Shake the plate at 1200 rpm for 10 seconds in the fluorescence plate reader to mix all of the components.
  7. Immediately start reading samples with an excitation wavelength of 488 nm and an emission wavelength of 515 nm. Samples should be read every minute for a total of 45 minutes.

Package Inserts

Package Inserts

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    Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.

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