Amplite® Colorimetric Peroxidase (HRP) Assay Kit Blue Color

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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Peroxidase is a small molecule (MW ~40 KD) that can usually be conjugated to an antibody in a 4:1 ratio. Due to its small size, it rarely causes steric hindrance problem with antibody/antigen complex formation. Peroxidase is inexpensive compared to other labeling enzymes. The major disadvantage associated with peroxidase is their low tolerance to many preservatives such as sodium azide that inactivates peroxidase activity even at low concentration. HRP conjugates are extensively used as secondary detection reagents in ELISAs, immuno-histochemical techniques and Northern, Southern and Western blot analyses. We offer this quick (10 min) HRP assay in a one-step, homogeneous, no wash assay system. This kit uses Amplite® Blue, our ultrasensitive chromogenic HRP substrate. Our Amplite® Blue is a chromogenic peroxidase substrate that is much more sensitive to both H2O2 and peroxidase than other chromogenic peroxidase substrates such as TMB, ABTS, OPD and K-Blue. Amplite® Blue generates a highly absorptive material that has maximum absorption of 664 nm. This near infrared absorption minimizes the background absorption that is often caused by the autoabsorption of biological samples that rarely absorb light beyond 600 nm. The kit can be used for ELISAs, characterizing kinetics of enzyme reaction and high throughput screening of oxidase inhibitors, etc. The kit provides an optimized 'mix and read' assay protocol that is compatible with HTS liquid handling instruments.

Platform

Absorbance microplate reader

Absorbance	664 ± 5 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol Summary

Prepare HRP standards and/or test samples (50 µL)
Add HRP working solution (50 µL)
Incubate at room temperature for 10 - 30 minutes
Monitor absorbance at 664 ± 5 nm

Important Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Blue Peroxidase Substrate stock solution (100X)

Add 250 µL of DMSO (Component E) into the vial of Amplite™ Blue Substrate (Component A) to make 100X Amplite^TM Blue Peroxidase Substrate stock solution.

2. HRP standard solution (20 U/mL)

Add 1 mL of Assay Buffer (Component C) into the vial of HRP (Component D) to make 20 U/mL HRP stock solution.

3. H₂O₂ solution (20 mM)

Add 22.7 µL of 3% H₂O₂ (0.88 M, Component B) into 977 µL of Assay Buffer (Component C) to make 20 mM H₂O₂ solution. Note: The diluted H₂O₂ solution is not stable. The unused portion should be discarded.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11551

HRP standard

Add 15 µL of 20 U/mL HRP solution into 985 µL of Assay Buffer (Component C) to get 300 mU/mL HRP standard solution (SD7). Take 300 mU/mL HRP standard solution (SD7) and perform 1:3 serial dilutions to get serially diluted HRP standards (SD6 - SD1) with Assay Buffer (Component C).

PREPARATION OF WORKING SOLUTION

Add 50 μL of Amplite™ Blue Peroxidase Substrate stock solution (100X) and 50 μL of H₂O₂ stock solution (20 mM) into 4.9 mL of Assay Buffer (Component C) to make a total volume of 5 mL HRP working solution. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of HRP standards and test samples in a white wall/clear bottom 96-well microplate. SD=HRP Standards (SD1 - SD7, 0.3 to 300 mU/mL); BL=Blank Control; TS=Test Samples.

BL	BL	TS	TS
SD1	SD1	...	...
SD2	SD2	...	...
SD3	SD3
SD4	SD4
SD5	SD5
SD6	SD6
SD7	SD7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
SD1 - SD7	50 µL	Serial Dilution (0.3 to 300 mU/mL)
BL	50 µL	Assay Buffer (Component C)
TS	50 µL	test sample

Prepare HRP standards (SD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of HRP working solution to each well of HRP standard, blank control, and test samples to make the total HRP assay volume of 100 µL/well. For a 384-well plate, add 25 µL of HRP working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 30 to 60 minutes, protected from light.
Monitor the absorbance with an absorbance plate reader at 664 ± 5 nm.

Product Family

Name	Excitation (nm)	Emission (nm)
Amplite® Fluorimetric Peroxidase (HRP) Assay Kit Red Fluorescence	571	584
Amplite® Fluorimetric Peroxidase (HRP) Assay Kit Near Infrared Fluorescence	648	668

Images

Figure 1. HRP dose response was measured with Amplite® Colorimetric Peroxidase Assay Kit in a white wall/clear bottom 96-well plate using a NovoStar absorbance microplate reader (BMG Labtech).

References

View all 49 references: Citation Explorer

Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots
Authors: Didenko VV, Baskin DS.
Journal: Biotechniques (2006): 295

Enzymatic oxidation of dipyridamole in homogeneous and micellar solutions in the horseradish peroxidase-hydrogen peroxide system
Authors: Almeida LE, Imasato H, Tabak M.
Journal: Biochim Biophys Acta (2006): 216

Recent advances in catalytic peroxidase histochemistry
Authors: Krieg R, Halbhuber KJ.
Journal: Cell Mol Biol (Noisy-le-grand) (2003): 547

Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells
Authors: Matsui T, Nakayama H, Yoshida K, Shinmyo A.
Journal: Appl Microbiol Biotechnol (2003): 517

Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization
Authors: van Gijlswijk RP, van de Corput MP, Bezrookove V, Wiegant J, Tanke HJ, Raap AK.
Journal: Histochem Cell Biol (2000): 175

Preparation, morphological characterization, and activity of thin films of horseradish peroxidase
Authors: Vianello F, Zennaro L, Di Paolo ML, Rigo A, Malacarne C, Scarpa M.
Journal: Biotechnol Bioeng (2000): 488

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid
Authors: Greco O, Folkes LK, Wardman P, Tozer GM, Dachs GU.
Journal: Cancer Gene Ther (2000): 1414

In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes
Authors: Schonhuber W, Zarda B, Eix S, Rippka R, Herdman M, Ludwig W, Amann R.
Journal: Appl Environ Microbiol (1999): 1259

Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements
Authors: Rota C, Chignell CF, Mason RP.
Journal: Free Radic Biol Med (1999): 873

Relative number of cells projecting from contralateral and ipsilateral nucleus isthmi to loci in the optic tectum is dependent on visuotopic location: horseradish peroxidase study in the leopard frog
Authors: Dudkin EA, Gruberg ER.
Journal: J Comp Neurol (1999): 212