Amplite® Fluorimetric Peroxidase (HRP) Assay Kit Near Infrared Fluorescence

Name: Amplite® Fluorimetric Peroxidase (HRP) Assay Kit *Near Infrared Fluorescence*
Brand: AAT Bioquest
SKU: 11553
Price: 341 USD
Availability: InStock

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® IR, our near infrared flurogenic HRP substrate. Amplite® IR generates a substance that has maximum absorption of 647 nm with maximum emission at 670 nm. This near infrared absorption and fluorescence minimize the assay background that is often caused by the autoabsorption and/or autofluorescence 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.

Example protocol

AT A GLANCE

Protocol Summary

Prepare HRP standards and/or test samples (50 µL)
Add Peroxidase working solution (50 µL)
Incubate at room temperature for 30 - 60 minutes
Monitor fluorescence intensity at Ex/Em = 640/680 nm (Cutoff = 665 nm)

Important Thaw all the kit components at room temperature before starting the experiment. The component A is unstable in the presence of thiols such as DTT and β-mercaptoethanol. The presence of thiols at concentration higher than 10 µM would significantly decrease the assay dynamic range. NADH and glutathione (reduced form: GSH) may interfere with the assay.

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™ IR Peroxidase Substrate stock solution (100X)

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

2. HRP standard solution (20 U/mL)

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

3. H₂O₂ stock 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 stock 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/11553

HRP standard

Add 15 µL of 20 U/mL HRP standard 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 100X Amplite™ IR Peroxidase Substrate stock solution and 50 μL of 20 mM H₂O₂ stock solution into 4.9 mL of Assay Buffer (Component C) to make Peroxidase working solution. Keep from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of HRP standards and test samples in a solid black 96-well microplate. SD= HRP Standards (SD1 - SD7, 0.41 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 Dilutions (0.41 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 Peroxidase working solution to each well of HRP standard, blank control, and test samples to make the total Peroxidase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Peroxidase 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 fluorescence increase with a fluorescence plate reader at Excitation = 600 - 650 nm, Emission = 650 - 690 nm (optimal Ex/Em = 640/680 nm, Cutoff = 665 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 647 ± 5 nm. The absorption detection has lower sensitivity compared to fluorescence reading.

Spectrum

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Product family

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

Citations

View all 1 citations: Citation Explorer

Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms
Authors: Li, Shuaizhang and Huang, Ruili and Solomon, Samuel and Liu, Yitong and Zhao, Bin and Santillo, Michael F and Xia, Menghang
Journal: Biotechnology Journal (2017): 1600715

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

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