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Amplite® Colorimetric Hydrogen Peroxide Assay Kit

Hydrogen Peroxide dose response was measured in a a white wall/clear bottom 96-well plate with the Amplite® Colorimetric Hydrogen Peroxide Assay Kit using a Spectramax absorbance microplate reader (Molecular Devices).
Hydrogen Peroxide dose response was measured in a a white wall/clear bottom 96-well plate with the Amplite® Colorimetric Hydrogen Peroxide Assay Kit using a Spectramax absorbance microplate reader (Molecular Devices).
Hydrogen Peroxide dose response was measured in a a white wall/clear bottom 96-well plate with the Amplite® Colorimetric Hydrogen Peroxide Assay Kit using a Spectramax absorbance microplate reader (Molecular Devices).
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Hydrogen peroxide (H2O2) is a reactive oxygen metabolic by-product that serves as a key regulator for a number of oxidative stress-related states. It is involved in a number of biological events that have been linked to asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, a number of neurodegenerative diseases and Down's syndrome. Perhaps the most intriguing aspect of H2O2 biology is the recent report that antibodies have the capacity to convert molecular oxygen into hydrogen peroxide to contribute to the normal recognition and destruction processes of the immune system. Measurement of this reactive species will help to determine how oxidative stress modulates varied intracellular pathways. This Amplite® Colorimetric Hydrogen Peroxide Assay Kit uses our unique Amplite® IR peroxidase substrate to quantify hydrogen peroxide in solutions and cell extracts. Upon hydrogen peroxide oxidation the colorless Amplite® IR generates an intense blue color product that is pH-independent from pH 4 to 10. The existing colorimetric hydrogen peroxide assays (from other vendors) often have severe sample interferences caused by the inherent absorption of biological samples. The near infrared absorption of Amplite® IR product minimizes the assay background that since the biological samples rarely absorb light beyond 600 nm. It can also be used to detect a variety of oxidase activities through enzyme-coupled reactions. The kit is an optimized 'mix and read' assay that is compatible with HTS liquid handling instruments.

Platform


Absorbance microplate reader

Absorbance650 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare H2O2 working solution (50 µL)
  2. Add H2O2 standards or test samples (50 µL)
  3. Incubate at room temperature for 10 - 60 minutes
  4. Monitor absorbance at 650 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment. 

Important notes
Amplite™ IR Peroxidase Substrate (Component A) is unstable in the presence of thiols such as DTT and β-mercaptoethanol. If the final concentration of the thiols is higher than 10 uM, it would significantly decrease the assay dynamic range. 

Important notes
NADH and glutathione (reduced form of GSH) may interfere with the assay.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ IR Peroxidase Substrate stock solution (100X):
Add 250 µL of DMSO (Component E) into the vial of Amplite™ IR Peroxidase Substrate (Component A).

2. Peroxidase stock solution (20 U/mL):
Add 1 mL of Assay Buffer (Component C) into the vial of Horseradish Peroxidase (Component D).

3. H2O2 standard solution (20 mM):
Add 22.7 µL of 3% H2O2 (0.88 M, Component B) into 977 µL of Assay Buffer (Component C). Note: The diluted H2O2 stock solution is not stable. The unused portion should be discarded.

PREPARATION OF STANDARD SOLUTION

H2O2 standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11500

Add 5 µL of 20 mM H2O2 standard solution into 995 µL of Assay Buffer (Component C) to get 100 µM H2O2 standard (HS7). Take 200 µL of 100 µM H2O2 standard to perform 1:2 serial dilutions to get serial dilutions of H2O2 standard (HS6 - HS1).

PREPARATION OF WORKING SOLUTION

Add 50 μL of Amplite™ IR Peroxidase Substrate Stock Solution (100X) and 200 μL of Peroxidase Stock Solution (20 U/mL) into 4.75 mL of Assay Buffer (Component C) to make a total volume of 5 mL. Note: Keep from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of H2O2 standards and test samples in a white wall/clear bottom 96-well microplate. HS= H2O2 Standards (HS1 - HS7, 1.563 to 100 µM); BL=Blank Control; TS=Test Samples.

BLBLTSTS
HS1HS1......
HS2HS2......
HS3HS3  
HS4HS4  
HS5HS5  
HS6HS6  
HS7HS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
HS1 - HS750 µLSerial Dilution (1.563 to 100 µM)
BL50 µLAssay Buffer (Component B)
TS50 µLtest sample

H2O2 assay in supernatants reaction

  1. Prepare H2O2 standards (HS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.

  2. Add 50 µL of H2O2 working solution to each well of H2O2 standard, blank control, and test samples to make the total H2O2 assay volume of 100 µL/well. For a 384-well plate, add 25 µL of H2O2 working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction at room temperature for 10 to 30 minutes, protected from light.

  4. Monitor the absorbance with an absorbance plate reader at 650 nm.

H2O2 assay for cells
The Amplite™ Colorimetric Hydrogen Peroxide Assay Kit can be used to measure the release of H2O2 from cells. The following is a suggested protocol that can be modified to meet the specific research needs.

  1. The H2O2 cell working solution should be prepared as given except that the Assay Buffer (Component C) should be replaced with the media used in your cell culture system. Suggested media including (a) Krebs Ringers Phosphate Buffer (KRPB); (b). Hanks Balanced Salt Solution (HBSS); or (c) Serum-free media.

  2. Prepare cells in a 96-well plate (50 - 100 µL/well), and activate the cells as desired. Note: The negative controls (media alone and non-activated cells) are included for measuring the background fluorescence.

  3. Add 50 µL of H2O2 cell working solution to each well of cells and H2O2 standards to make the total H2O2 assay volume of 100 µL/well. For a 384-well plate, add 25 µL of H2O2 cell working solution into each well instead, for a total volume of 50 µL/well.

  4. Incubate the reaction at room temperature for 10 to 60 minutes, protected from light.

  5. Monitor the absorbance with an absorbance plate reader at 650 nm.

Images


Citations


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Mild Oxidative Stress Reduces NRF2 SUMOylation to Promote Kras/Lkb1/Keap1 Mutant Lung Adenocarcinoma Cell Migration and Invasion
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References


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Genetically encoded fluorescent indicator for intracellular hydrogen peroxide
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Journal: Nat Methods (2006): 281
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Journal: Cell Biol Toxicol (2006): 39
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Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo
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