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Amplite® Colorimetric Antioxidant Activity Assay Kit
Example protocol
AT A GLANCE
Protocol summary
- Prepare samples/Trolox (10 µL)
- Add HRP working solution (20 µL)
- Add ReadiUse™ ABTS Substrate Solution (150 µL)
- Incubate at room temperature for 5 minutes
- Monitor Absorbance at 405 nm
Important notes
Equilibrate all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Horseradish Peroxidase (HRP) stock solution (2000X):
Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component A).
2. Trolox standard solution (100 mM):
Add 100 µL of DMSO into the vial of Trolox (Component D).
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15900
Prepare a Trolox standard by diluting 100 mM Trolox standard solution in assay buffer (Componenet B) using dilution factor =2 and the top concentration = 1 mM.
PREPARATION OF WORKING SOLUTION
HRP working solution (1X):
Add 2.5 uL of 2000X HRP stock solution into 5 mL of Assay Buffer (Component B) to make 1X HRP working solution.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of Trolox standards and test samples in a clear 96-well microplate. SD = trolox standard (SD1 - SD7, 1 to 0.0156 mM), BL = blank control, TS = test sample.
| 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 | 10 µL | serial dilution (1 to 0.0156 mM) |
| BL | 10 µL | Assay Buffer (Component B) |
| TS | 10 µL | sample |
- Prepare trolox standards (SD), blank controls (BL), and test samples (TS) (dilute the samples in Assay Buffer to bring the antioxidant level within the same range as Trolox, if necessary) according to the layout provided in Table 1 and Table 2.
- Add 20 µL of HRP working solution into each well of Trolox standards, blank control, and test samples to make the total volume of 30 µL/well.
- Add 150 µL of ReadiUse™ ABTS Substrate Solution (Component C) to each well, for a total volume of 180 µL/well. Note: Avoid light and use in polypropylene containers.
- Incubate for 5 minutes at room temperature. Note: This 5 minutes incubation time is a guideline. The incubation time can be changed to obtain a more suitable absorbance.
- Read the absorbance at 405 nm using an absorbance microplate reader.
Safety data sheet