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Amplite® Luminometric Peroxidase (HRP) Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare HRP working solution (50 µL)
- Add HRP standards and/or test samples (50 µL)
- Incubate at room temperature for 30 minutes to 2 hours
- Monitor luminescent intensity
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
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
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.
| BL | BL | TS | TS |
| PS1 | PS1 | ... | ... |
| PS2 | PS2 | ... | ... |
| PS3 | PS3 | ||
| PS4 | PS4 | ||
| PS5 | PS5 | ||
| PS6 | PS6 | ||
| PS7 | PS7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| PS1 - PS7 | 50 µL | Serial Dilution (0.156 to 10 mU/mL) |
| BL | 50 µL | PBS with 0.1% BSA |
| TS | 50 µL | test sample |
- 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.
- 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 minutes to 2 hours, protected from light.
- Monitor the luminescence intensity by using a standard luminometer.
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 |
References
Authors: Didenko VV, Baskin DS.
Journal: Biotechniques (2006): 295
Authors: Almeida LE, Imasato H, Tabak M.
Journal: Biochim Biophys Acta (2006): 216
Authors: Krieg R, Halbhuber KJ.
Journal: Cell Mol Biol (Noisy-le-grand) (2003): 547
Authors: Matsui T, Nakayama H, Yoshida K, Shinmyo A.
Journal: Appl Microbiol Biotechnol (2003): 517
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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