Hydrogen Peroxide Assay Kit (ab102500)

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.

As recommended on the protocol, an amount ranging from 2 to 50ul should be added to each well, and fill it up to 50ul with assay buffer.

As the concentration of H2O2 in your samples is unknown, you may need to run a quick test beforehand to optimise the volume of sample per well.

As an example, this kit was used on reference PubMed: 24187462, in which 5 μL of the rat sample supernatant was diluted with 46 μL of assay buffer.

The kit ab102500 is suitable with many type of biological fluids and extracts such as cell lysates and tissue culture supernatant.

Frozen sample can be used with this kit so once the extract is prepared these should be immediately frozen at -20C or -80C for later use. We see people successfully 1-2 year frozen lysates so we unfortunately will not be able to advice any time limit. I would say for better results storage for couple of month should be fine.

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Since we mention only a 10 min incubation time for this reaction, I would recommend taking the reading right after. Waiting for more time might result in increase in the background signal and later drop in the total signals.
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Please buy the requested product ab167452.

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I have sent a request to my colleague for adding this product in catalogue. I will let you know the catalogue number soon.

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We can provide one bottle of assay buffer for £90. Please let us know if you are interested in buying.

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This kit can actually be used both colorimetrically and fluorometrically. In general fluorometric analysis is ˜10 times more sensitive than colorimetric analysis. The detection limit with this assay can be as low as 2 pmol/well (or 40 nM) of H2O2 in the sensitive fluorometric assay.
As long as the H2O2 concentration is higher than that in the skeletal muscle samples, you would be able to use this kit successfully.
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We recommend the use of the 10 kDa filters irrespective of your immediate or later usage of the lysed samples. The filters help in separating out proteins which can chew up the analyte. You most likely will not get efficient results without the fliters.

We carry 10 kD spin columns to assist in using this kit. The link for these columns is given below:
https://www.abcam.com/10kd-spin-column-ab93349.html

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You can use the H2O2 assay buffer in the kit as the lysis buffer for cells.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.