Peroxidase Activity Assay Kit 产品说明书

Technical Bulletin

Peroxidase Activity Assay Kit

Catalog Number MAK092

Product Description

Peroxidase is an enzyme found broadly in biological

systems that utilizes hydrogen peroxide in the

oxidation of various substrates.

The Peroxidase Activity Assay Kit provides a simple

and direct procedure for measuring peroxidase

activity in a variety of biological samples. Peroxidase

catalyzes the reaction between H

2

O

2

and the probe,

resulting in a colorimetric (570 nm) or fluorometric

(

Ex

= 535/

Em

= 587 nm) product proportional to the

peroxidase activity present. One unit of peroxidase is

defined as the amount of enzyme that reduces

1.0 µmole of H

2

O

2

per minute at 37 C.

The kit is suitable for the determination of peroxidase

activity in cell culture supernatant, serum, plasma,

urine, and other biological fluids.

Components

The kit is sufficient for 100 colorimetric or

fluorometric assays in 96-well plates.

 Assay Buffer 25 mL

Catalog Number MAK092A

 Fluorescent Peroxidase 0.2 mL

 Substrate, in DMSO

Catalog Number MAK092B

 H

2

O

2

Substrate, 0.88 M 0.1 mL

Catalog Number MAK092C

 HRP Positive Control 1 vial

Catalog Number MAK092D

Document MAK092 Rev 09/22

Equipment Required but Not Provided

 Pipetting devices and accessories (e.g.,

multichannel pipettor)

 Fluorescence or spectrophotometric multiwell

plate reader

 96-well plates. It is recommended to use black

plates with clear bottoms for fluorescence assays

and clear plates for colorimetric assays. Cell

culture or tissue culture treated plates are not

recommended.

 Dounce tissue grinder set

(Catalog Number D9063 or equivalent)

 Refrigerated microcentrifuge capable of

RCF ≥ 1,000 × g

Precautions and Disclaimer

For R&D use only. Not for drug, household, or other

uses. Please consult the Safety Data Sheet for

information regarding hazards and safe handling

practices.

Storage/Stability

The kit is shipped on wet ice. Store components

at -20 °C, protected from light.

Preparation Instructions

Briefly centrifuge small vials prior to opening.

Assay Buffer: Bring to room temperature prior to use.

Fluorescent Peroxidase Substrate: Prior to use, briefly

warm at 37 °C for 1-2 minutes to completely melt

DMSO solution, mix well. Store at –20 °C.

H

2

O

2

Substrate: See Standard Curve Preparation

Procedure.

1

HRP Positive Control: Reconstitute the vial with 1 mL

of Assay Buffer. The reconstituted HRP Positive

Control solution is stable for one day at 2-8 °C and

one month at -20 °C. Keep the solution on ice while

in use.

Procedure

Sample Preparation

Both the colorimetric and fluorometric assays require

50 µL of sample for each reaction (well).

1. Collect cell culture supernatant, serum, plasma,

urine, and/or other biological fluids.

2. Centrifuge test samples for 15 minutes at

1,000 × g within 30 minutes of collection to

remove insoluble materials. Retain supernatant

for assay. Assay immediately or aliquot and store

the samples at -80 °C. Avoid repeated

freeze-thaw cycles.

3. Add 2-50 µL of Sample (S) into desired well(s) in

96-well plate. For unknown samples, test different

amounts of Sample to ensure the readings are

within the Standard Curve range.

4. Adjust the total volume of each Sample (S) well

to 50 L with Assay Buffer.

HRP Positive Control Preparation

1. Dilute HRP Positive Control 1:199 in Assay Buffer.

2. Add 1 µL of diluted Positive Control into desired

wells in 96-well plate.

3. Adjust the total volume of each Positive Control

well to 50 µL by adding 49 µL of Assay Buffer.

Colorimetric Standard Curve Preparation

1. Prepare a 12.5 mM H

2

O

2

Substrate solution by

mixing 5 µL of the 0.88 M H

2

O

2

Substrate with

347 L of Assay Buffer. Mix well by pipetting (do

not vortex), then aliquot and store at -20 C.

The diluted H

2

O

2

Substrate is stable for one day

at 2-8 °C and one month at -20 °C.

2. Prepare a 0.1 mM H

2

O

2

Substrate Solution by

mixing 10 µL of the 12.5 mM H

2

O

2

Substrate

solution with 1240 µL of Assay Buffer.

Document MAK092 Rev 09/22

3. Prepare H

2

O

2

Standards in separate wells of the

96-well plate according to Table 1.

Table 1.

Preparation of Colorimetric H

2

O

2

Standards

0.1 mM

Well

H

2

O

2

Assay H

2

O

2

Substrate Buffer

(nmol/well)

Solution

1 - 50 µL 0

2 10 µL 40 µL 1

3 20 µL 30 µL 2

4 30 µL 20 µL 3

5 40 µL 10 µL 4

6 50 µL - 5

Fluorometric Standard Curve Preparation

1. Prepare a 0.1 mM H

2

O

2

Substrate solution as

directed in Steps 1 and 2 of the Colorimetric

Standard Curve Preparation Procedure.

2. Prepare a 0.01 mM H

2

O

2

Substrate solution by

mixing 100 µL of the 0.1 mM H

2

O

2

Substrate

solution with 900 µL of Assay Buffer.

3. Prepare H

2

O

2

Standards in separate wells of the

96-well plate according to Table 2.

Table 2.

Preparation of Fluorometric H

2

O

2

Standards

0.01 mM

Well

H

2

O

2

Assay H

2

O

2

Substrate Buffer

(pmol/well)

Solution

1 - 50 µL 0

2 10 µL 40 µL 100

3 20 µL 30 µL 200

4 30 µL 20 µL 300

5 40 µL 10 µL 400

6 50 µL - 500

2

Standard Curve Reaction Mix

1. Dilute HRP Positive Control 1:199 in Assay Buffer.

2. Mix enough reagents for the number of assays to

be performed. For each Standard well, prepare

50 µL of Standard Curve Reaction Mix according

to Table 3.

Table 3.

Preparation of Standard Curve Reaction Mix

Reagent Volume

Fluorescent Peroxidase

Substrate

2 µL

Diluted HRP Positive Control 48 µL

3. Mix well and add 50 µL of Standard Curve

Reaction Mix to each Standard well.

4. Mix well and incubate at room temperature for

5 minutes.

5. For colorimetric assay, measure the absorbance

at 570 nm (A). For fluorometric assay, measure

fluorescence intensity (RFU) at



Ex

= 535/

Em

= 587 nm.

Sample and HRP Positive Control Reaction Mix

1. Mix enough reagents for the number of assays to

be performed. For each Sample and HRP Positive

Control well, prepare 50 µL of Sample and

HRP Positive Control Reaction Mix according

to Table 4.

Table 4.

Preparation of Sample and HRP Positive Control

Reaction Mix

Reagent Volume

Assay Buffer 46 µL

Fluorescent Peroxidase

Substrate

2 µL

12.5 mM H

2

O

2

Substrate 2 µL

2. Mix well and add 50 µL of Sample and HRP

Positive Control Reaction Mix to each Sample and

HRP Positive Control well.

3. Mix well.

4. Incubate the plate at 37 C. After 3 minutes, take

the initial measurement. For colorimetric assays,

measure the absorbance at 570 nm (A

Initial

). For

fluorometric assays, measure fluorescence

intensity (RFU

Initial

) at 

Ex

= 535/

Em

= 587 nm).

Document MAK092 Rev 09/22

5. Incubate the plate at 37 C for another

30 minutes to 2 hours. Incubation time will

depend on the peroxidase activity in the samples.

Protect the plate from light during the incubation.

6. For colorimetric assays, measure the absorbance

(A

Final

) after incubation is complete. For

fluorometric assays, measure RFU

Final

after

incubation is complete.

7. Alternatively, take measurements every

3-5 minutes in kinetic method and choose the

period of linear range which falls within the

H

2

O

2

Standard Curve to calculate the peroxidase

activity of the Samples. It is essential that the

final measurement falls within the linear range of

the standard curve.

Results

1. Use optical density (A) or fluorescence intensity

(RFU) measured in Step 5 of the Standard Curve

Reaction Mix Procedure.

2. Determine ΔOD or ΔF by subtracting the Blank

value (Standard #1) from the remaining

standard values.

3. Plot the ΔOD or ΔF against standard

concentrations and determine the slope of the

H

2

O

2

Standard Curve by linear regression.

4. Calculate the change in absorbance (ΔA) or

fluorescence intensity (ΔRFU) for each sample:

ΔA = A

Final

– A

Initial

or

ΔRFU = RFU

Final

– RFU

Initial

5. Apply the ΔA or ΔRFU to the H

2

O

2

Standard Curve

to get B nmol of H

2

O

2

generated by peroxidase in

the given reaction time.

Peroxidase Activity (nmol/min/mL or mU/mL) =

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