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Peroxidase assay


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#1 giny

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Posted 09 February 2010 - 02:21 AM

Hi,

I am looking for a peroxidase assay kit. I want to quantify the activity of a peroxiredoxin in my experiment. Does anyone of you know any good peroxidase assay kit? Please let me know. Thank you

#2 Zagami Francesco

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Posted 08 July 2019 - 11:38 PM

Aminoantipyrine assay for research peroxidase activity in the stool

Principle

Feces components having peroxidase activity, such as haemoglobin substance, and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to aminoantipyrine. Oxidation of the aminoantipyrine produces a pink color whose intensity is proportional to the concentration of peroxidase activity contained in the stool specimen.

Reagents

AAP reagent (30 ml): dissolve 0.8 g aminoantipyrine in 30 ml 95% ethanol and place in amber glass bottle strictly closed. Stable 1 year at 4 °C.

Acid solution (2.0 ml) : dilute 0.5 ml acetic acid to 5 ml with dH2O, and placed in clear plastic. Stable 1 year at 4 °C.

Peroxide 3% - 10 Vol. (2.0 ml): dilute 170 µl of 35% hydrogen peroxide stock solution, in 2.0 ml of phosphate buffer 40 mM at pH 7.0 (dissolve 23.2 mM Na2HPO4•2H2O, and 16.8 mM NaH2PO4•2H2O) containing 1 g/L ethylenediaminetetraacetic acid (EDTA), check pH and adjust it to 7.0 if necessary using HCl or NaOH, and store in opaque plastic at 4 °C, resulting stable 1 year.

Working Solution (WS): Immediately before use mix 4.5 ml of AAP reagent with 0.3 ml of acid solution.

Sample preparation and assay: dispense in container stool collection, a volumes of dH2O having ratio 7:1 with feces contents (e.g. 1 g feces, and 7 ml of dH2O) and to emulsify it using a glass stick.  Centrifuge 2.5 ml of emulsified specimen at 10900 rpm for 15 min. and then dispense 1.4 ml of supernatant in transparent glass tube, while for positive control, dispense 1.4 ml of dH2O containing 50 µl of pre-diluted blood (5 µl of whole blood EDTA-K3 in 1 ml of dH2O), and for blank 1.4 ml of dH2O. Layer, in each tube,1.4 ml of WS on top sample, avoiding mix both solutions. After add 0.280 ml of peroxide, into WS, avoiding always, of mix the sample solution and WS, allow to stand for 1 min. Look the contact point between WS and sample or positive control and verify the possible appearance of a layer of pink color. In negative peroxidase activity, this should show no color change, while in positive control this should show a positive reaction (Fig. 1) from pale pink (+) across pink dark (++) to pink back  (+++)

 

 






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