Hydrogen Peroxide Colorimetric/Fluorometric Assay Kit

Sensitive Assay, HTS
Catalog #: K265 | abID: ab102500

Product Details

abID ab102500
Cat # +Size K265-200
Size 200 assays
Detection Method Absorbance (570 nm)and fluorescence (Ex/Em 535/587 nm)
Species Reactivity Mammalian
Applications The kit provides a highly sensitive, simple, direct and HTS-ready colorimetric assay for measuring H₂O₂ in biological samples.
Features & Benefits • Simple procedure; takes less than ~1 hours
• Fast and convenient
• The kit can perform 200 reactions by fluorometric method or 100 reactions by colorimetric method. The detection limit can be as low as 2 pmol per assay (or 40 nM concentration) of H₂O₂ in the sensitive fluorometric assay.
Kit Components • H₂O₂ Assay Buffer
• OxiRed™ Probe
• Dimethylsulfoxide (DMSO, anhydrous)
• H₂O₂ Standard (0.88M)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Hydrogen Peroxide is a reactive oxygen metabolic byproduct that serves as a key regulator for a number of oxidative stress-related states. Functioning through NF-κB and other factors, hydroperoxide-mediated pathways have been linked to asthma, inflammatory arthritis, atherosclerosis, diabetic vasculopathy, osteoporosis, neuro-degenerative diseases, Down’s syndrome and immune system diseases. BioVision’s Hydrogen Peroxide Assay Kit is a highly sensitive, simple, direct and HTS-ready colorimetric and fluorometric assay for measuring H₂O₂ in biological samples. In the presence of Horse Radish Peroxidase (HRP), the OxiRed Probe reacts with H₂O₂ to produce product with color (λmax = 570 nm) and red-fluorescent (Ex/Em=535/587 nm). The kit can perform 200 reactions by fluorometric method or 100 reactions by colorimetric method. The detection limit can be as low as 2 pmol per assay (or 40 nM concentration) of H₂O₂ in the sensitive fluorometric assay.

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Could you please specify how to prepare the samples? How should the tissue be homogenized (in which liquid) and how many milligrams should be used?
They can start with 50 mg tissue and homogenize in 300 - 400 µl of the Assay Buffer. Homogenization can be done using a dounce homogenizer on ice or mild sonication (few short pulses). During homogenization, samples should be kept on ice. Following homogenization, tissue lysate should be centrifuged at 10,000xg for 2 min.Collect the supernatant and filter through a 10 kDa MW spin column (BV Cat. # 1997).
Will this kit detect other peroxyl groups such as Ros or superoxide?
No, this kit is specific for hydrogen peroxide.
Can the colour be read at 540nm?
Most plate readers have flexibility in their band width of detection. Your wavelength of 540 nm for a recommended wavelength of 570 nm should work perfectly fine as long as it has the flexibility of +/- 30 nm.
How does the colorimetric sensitivity of this kit compare with the fluorometric version?
The colorimetric method is in general ~10 times less sensitive than the fluorometric one.
We froze cells at -80C prior to lysis. Following lysis, if 10kDa filter is not used, is it ok to freeze samples?
We would recommend the use of the 10 kDa filters irrespective of your immediate or later usage of the lysed samples. The filters wills help in separating out proteins which can chew up the analyte.
Could you please confirm if we could use different wells for samples on different days? So could we perform 4 measurements each on following days or would you expect any complications?
If you want to use the same plate but different wells on consecutive days, you can do that, provided you are not directly comparing the readings between them.
Can we use this kit for tissue samples? Do we need to deproteinize?
Yes, you can most definitely use this kit with tissue samples. Tissue lysates should be filtered through a 10 kDa MW spin filter (BioVision, Cat 1997-25) to remove all proteins.
I have a question about the standards: Do we need to make up the volume to 50ul for each standard? If we need to make up the volume to 50 do I use the assay buffer or dH2O?
Yes, please make up the standards to 50 µl with the assay buffer.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, we would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Hey-Min Kim, et al., (2018) Stimulation of Vibrio vulnificus Pyruvate Kinase in the Presence of Glucose to Cope With H2O2 Stress Generated by Its Competito, Frontiers in Microbiology, May 2018, 29896177
Erin K. Zinkhan, et al., (2018) Prenatal Exposure to a Maternal High Fat Diet Increases Hepatic Cholesterol Accumulation in Intrauterine Growth Restricted Rats in Part Through MicroRNA-122 Inhibition of Cyp7a1, Frontiers in Physiology, May 2018, 29896121
Lee, Y. H. et al., (2017) Nanoparticle mediated PPARγ gene delivery on dental implants improves osseointegration via mitochondrial biogenesis in diabetes mellitus rat model, Nanomedicine: Nanotechnology, Biology and Medicine, Jul.2017, https://doiorg/101016/jnano201702020
Bito, Tomohiro et al. (2017) Vitamin B12 deficiency results in severe oxidative stress, leading to memory retention impairment in Caenorhabditis elegans, Redox Biol. 2017 Apr;11:21-29.
Wang, Shun-Ping et al. (2016) Potent Antiarthritic Properties of Phloretin in Murine Collagen-Induced Arthritis, Evid Based Complement Alternat Med. 2016;2016:9831263.
For more citations of this product click here