Peroxidase Activity Assay Kit (BA0008)

Product Name:	Peroxidase Activity Assay Kit (BA0008)
SKU:	BA0008
Detection Method:	Colorimetric (OD 570 nm) / Fluorometric (Ex 530 / Em 585 nm)
Detection Range:	Colorimetric 2 to 50 U/L; fluorimetric 0.1 to 5 U/L
Sample Type:	['Plasma', 'Serum', 'Urine', 'Tissue', 'Culture media']
Species Reactivity:	All
Assay Time:	~20 minutes (10 min incubation plus stop)
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	Assay Buffer, Dye Reagent and Resorufin at -20°C; 3% Stabilized H2O2 at -20 to 4°C; Stop Reagent at any temperature between -20°C and room temperature
Shelf Life:	12 months after receipt
Shipping:	Room Temperature

Peroxidases (EC 1.11.1.x) catalyse oxidation-reduction reactions in which a peroxide is reduced while an electron donor is oxidised. For many peroxidases the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. In the cell, peroxidases destroy toxic hydroxide radicals formed as by-products during aerobic respiration. The peroxidases represent a large family of enzymes found in animals, plants, fungi and bacteria. This assay uses hydrogen peroxide and an electron-donor dye that forms resorufin during the peroxidase reaction; the optical density (570 nm) or fluorescence intensity (excitation 530 nm, emission 585 nm) is a direct measure of the enzyme activity.

Safe. Non-radioactive assay.
Sensitive and accurate. Use as little as 10 µL sample. Linear detection range: colorimetric assays 2 to 50 U/L, fluorimetric assays 0.1 to 5 U/L peroxidase.
Convenient and high-throughput. Mix-incubate-measure type assay that can be readily automated with HTS liquid handling systems.

Peroxidase activity determination in biological samples (e.g. plasma, serum, urine, tissue and culture media).

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Prepare samples according to established methods, testing multiple sample dilutions to ensure activity is in the linear range. Bring all reagents to room temperature and dilute the 3% H2O2 in Assay Buffer to 0.6%, using within one hour.
2	Colorimetric procedure: Transfer 100 µL H2O and 100 µL Resorufin into two wells of a clear flat-bottom 96-well plate. Transfer 10 µL H2O (sample blank) and 10 µL sample to separate wells. Prepare enough Working Reagent by mixing, for each well, 95 µL Assay Buffer, 0.5 µL Dye Reagent and 0.5 µL freshly diluted 0.6% H2O2, add 90 µL Working Reagent to each sample well, tap to mix and incubate for 10 min at room temperature.
3	Add 100 µL Stop Reagent to all wells, tap the plate to mix and read OD570nm.
4	Fluorimetric procedure (linear range 0.1 to 5 U/L): Dilute the Resorufin 1:10 in dH2O. Transfer 100 µL H2O and 100 µL diluted Resorufin into two wells of a black flat-bottom 96-well plate. Transfer 10 µL H2O and 10 µL sample to separate wells, add 90 µL Working Reagent to each well (prepared as above), tap to mix and incubate for 10 min at room temperature.
5	Add 100 µL Stop Reagent to all wells, tap the plate to mix and read fluorescence (excitation 530 nm, emission 585 nm).

Peroxidase Activity = [(RSample - RBlank) / (RResorufin - RH2O)] x [Resorufin] x n (U/L), where RSample, RBlank, RResorufin and RH2O are the OD or fluorescence readings of the Sample, Sample Blank, Resorufin and Water, and n is the dilution factor. The [Resorufin] is 50 µM for colorimetric assays and 5 µM for fluorimetric assays. If sample readings exceed those of the Resorufin, dilute the sample in Assay Buffer, repeat and multiply by n. Unit definition: one unit of enzyme catalyses the formation of 1 µmole resorufin per min under the assay conditions.

Component	Quantity	Storage
Assay Buffer	20 mL	-20°C
Dye Reagent	60 µL	-20°C
Resorufin	1.5 mL	-20°C
Stop Reagent	12 mL	-20°C to room temperature
3% Stabilized H2O2	100 µL	-20 to 4°C