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Amplite® Luminometric Peroxidase (HRP) Assay Kit

HRP dose response was measured with Amplite® Luminometric Peroxidase Assay Kit in a solid black 384-well plate using a NOVOstar plate reader (BMG Labtech).
HRP dose response was measured with Amplite® Luminometric Peroxidase Assay Kit in a solid black 384-well plate using a NOVOstar plate reader (BMG Labtech).
HRP dose response was measured with Amplite® Luminometric Peroxidase Assay Kit in a solid black 384-well plate using a NOVOstar plate reader (BMG Labtech).
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


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. 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


Luminescence microplate reader

Recommended plateSolid white

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare HRP working solution (50 µL)
  2. Add HRP standards and/or test samples (50 µL)
  3. Incubate at room temperature for 30 minutes to 2 hours
  4. Monitor luminescent intensity

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

PREPARATION OF STOCK SOLUTION

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. HRP stock solution (20 U/mL):
Add 1 mL of PBS with 0.1% BSA into the vial of Horseradish Peroxidase (Component C).

PREPARATION OF STANDARD SOLUTION

HRP standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11559

Add 1 μL of 20 U/mL HRP stock solution in 1999 μL of PBS with 0.1% BSA to get 10 mU/mL HRP standard solution (PS7). Then use 10 mU/mL standard solution and perform 1:2 serial dilutions to obtain remaining serially diluted standards (PS6-PS1).

PREPARATION OF WORKING SOLUTION

Add 30 μL of 3% stabilized H2O2 solution (Component B) into 5 mL of Assay Buffer (Component A) to make HRP working solution and keep from light. Note: The HRP working solution is stable at room temperature for at least 8 hours without activity loss if kept from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of HRP standards and test samples in a solid black 96-well microplate. PS=Peroxidase Standards (PS1-PS7, 0.156 to 10 mU/mL), BL=Blank Control, TS=Test Samples.

BLBLTSTS
PS1PS1......
PS2PS2......
PS3PS3  
PS4PS4  
PS5PS5  
PS6PS6  
PS7PS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
PS1 - PS750 µLSerial Dilution (0.156 to 10 mU/mL)
BL50 µLPBS with 0.1% BSA
TS50 µLtest sample
  1. Prepare HRP standards (PS), 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.

  2. 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.

  3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.

  4. Monitor the luminescence intensity by using a standard luminometer.

Images


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