Overview

  • Product name

    Xanthine Oxidase Activity Assay Kit
  • Detection method

    Colorimetric/Fluorometric
  • Sample type

    Cell culture supernatant, Milk, Urine, Serum, Plasma, Other biological fluids, Tissue Extracts
  • Assay type

    Enzyme activity
  • Sensitivity

    > 10 mU/ml
  • Range

    10 mU/ml - 1000 mU/ml
  • Assay time

    0h 40m
  • Product overview

    Xanthine Oxidase Activity Assay Kit ab102522 is an easy and sensitive assay to determine xanthine oxidase activity in variety of samples.


    In the xanthine oxidase assay protocol, xanthine oxidase oxidizes xanthine to hydrogen peroxide (H2O2) which reacts stoichiometrically with a probe to generate color (at OD = 570 nm) and fluorescence (at Ex/Em = 535/587 nm). Since the color or fluorescence intensity is proportional to XO content, the XO activity can be accurately measured.


    The kit detects 1-100 mU xanthine oxidase in 100 µl reaction volume.


    Xanthine oxidase assay protocol summary:
    - add samples and standards to wells
    - add reaction mix
    - analyze with microplate reader immediately and after 10-20 min

  • Notes

    Xanthine oxidase (XO, EC 1.17.3.2 ) is present in appreciable amounts in the liver and jejunum in healthy individuals. However, in various liver disorders, XO is released into circulation. Therefore, determination of serum XO level serves as a sensitive indicator of acute liver damage such as jaundice.

  • Platform

    Microplate reader

Properties

Images

  • Xanthine Oxidase Positive Control

Protocols

References

This product has been referenced in:

  • Kimura Y  et al. Canagliflozin, a sodium-glucose cotransporter 2 inhibitor, normalizes renal susceptibility to type 1 cardiorenal syndrome through reduction of renal oxidative stress in diabetic rats. J Diabetes Investig 10:933-946 (2019). Read more (PubMed: 30663266) »
  • Wert KJ  et al. Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface. Free Radic Biol Med 124:408-419 (2018). Read more (PubMed: 29940351) »
See all 8 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

It will be possible to prepare and use the ab102522 Xanthine Oxidase Assay Kit several times over two months.

Once you have opened the kit and prepared the components, it is highly recommended that the components be stored in aliquots at -20C.  Aliquoting will avoid freeze - thaw cycles.  The assay buffer can be stored at 4oC.  All components should be used within 2 months.


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Answer

"Cell culture supernatants" in this instance means cell lysates. Xanthine oxidase is not released by healthy cells. To prepare cell lysates, simply pellet the cells, estimate the volume of the pellet, and add 4 volumes of the assay buffer that is provided with the kit. The cells can be homogenized in the buffer and then centrifuged to pellet debris that does not go into solution. We recommend a Dounce homogenizer on ice. The sample to be assayed is the supernatant. These instructions are in step 1.b. of the assay protocol, which is linked to the online datasheet.

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Answer

I can confirm that the kit ab102522 and ab102500 can be used to assay xanthine oxidase and Hydrogen peroxide in cells. I would suggest starting with at least 1-2 million cells. Please follow the following protocol;

2X106 cells, suspend the cell pellet 500 μl (or ˜4 volumes) of the assay buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Load the supernatant unto a 10kda spin column for deproteinzation (if indicated in the protcol booklet). Use the eluate for your subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve.

These kits assay different biochemicals one is specific to XO and other one to H2O2. No doubt the same Oxi red probe is used in the kits as both are based on reaction between H2O2 and oxi red probe; this however doesn’t mean the kits are same.

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Answer


En cuanto al principio de la reacción, en el “enzyme mix” no hay enzima XO. Este reactivo actúa en el segundo paso de la reacción, después de que la enzima XO de las muestras haya degradado la Xantina. El enzyme mix cataliza la reacción mediante el “probe” y el peróxido de hidrogeno generado. El color desarrollado es proporcional a la cantidad de H2O2 producida por la reacción catalizada por XO.

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Answer

The cell pellet after removing all media can be stored at -80C with a small volume of PBS. It is not ideal to store cells with medium. Once thawed, the cells can be washed once with PBS and then the PBS can be removed. At this step the assay buffer can be used for extraction according to the protocol.

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Answer

I have double checked with the lab. Yes, the assay buffer is sufficient for efficient homogenization. You don’t need any other buffer. Just look at an aliquot of the homogenised sample under the microscope. If most cells show a broken nuclear ring, the homogenization is good, if not, you need to homogenize further.

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Answer

Thank you for your enquiry.

I am pleased to provide the following information for you:

1. Is there a preferred method to lift up the cells before centrifuging to get the supernatant/xanthine oxidase?

Cells can be trypsinized and washed with ice cold PBS before use with this assay.

2. What is the optimal number of cells to lift up per assay?

Use at least 10^6 cells for the assay.

3. How much volume of assay buffer should be added when centrifuging? Since my cells are cultured on a plate, it's hard to determine what "4 volumes" of assay buffer would be

Use 200 ul of the assay buffer with each 10^6 cells for homogenization.

4. Is it possible to perform the assay by using individual cuvettes and if so, what changes to the protocol should be followed?

This is a plate reader based method. We would not recommend using a cuvette as this will lead to different incubation times in different samples, so the results would not be comparable.


I hope this information will be helpful. If you have any further questions, please do not hesitate to contact me.

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Answer

Thanks for your inquiry.
You can most likely use frozen tissues with limitations. We ourselves have never tried it, but I think if the tissues have been frozen efficiently and stored well, they should not pose a problem. Unfortunately we cannot guarantee that it will work since the kit hasn't been validated with frozen tissues.
Here are a few references which might come in handy:

Ha, Y. et. al. Sigma Receptor 1 Modulates Endoplasmic Reticulum Stress in Retinal Neurons. Invest. Ophthalmol. Vis. Sci., Jan 2011; 52: 527 - 540.
Tual-Chalot, S. et al. Circulating Microparticles from Pulmonary Hypertensive rats Induce Endothelial Dysfunction. Am. J. Respir. Crit. Care Med., 2010; 182: 261-268.
Tual-Chalot, S. et al. Circulating Microparticles from Pulmonary Hypertensive Rats Induce Endothelial Dysfunction. Am. J. Respir. Crit. Care Med. 2010 10.1164/rccm.200909-1347OC
I hope this information helps. Please contact us with any other questions.

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