Amplite® Luminometric Peroxidase (HRP) Assay Kit
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | SDSProtocol |
Platform
Luminescence microplate reader
Recommended plate | Solid white |
Components
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 |
Images
References
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Authors: Dudkin EA, Gruberg ER.
Journal: J Comp Neurol (1999): 212
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