Amplite® Colorimetric Antioxidant Activity Assay Kit

Ordering information

Price
Catalog Number
Unit Size
Quantity

Add to cart

Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
International	See distributors
Bulk request	Inquire
Custom size	Inquire
Shipping	Standard overnight for United States, inquire for international

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Reactive oxygen species (ROS) are essential in cell signaling and maintaining physiological cell metabolism. An excessive level of ROS damages cellular components, which links to numerous diseases, including cancer, metabolic disorders, neurodegenerative disease, etc. Antioxidants, including enzymes, macromolecules, and small molecules, are generated to combat the ROS-induced damages in living organisms. Quantitative analysis of the antioxidant activity in body fluids, tissues, and cells provides important biological information. Our Amplite® Colorimetric Antioxidant Assay Kit uses ABTS as a colorimetric indicator of antioxidant activity based on the observation that the ability of antioxidants to prevent the oxidation of ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) to ABTS+ by Horseradish Peroxidase (HRP) and Hydrogen peroxide (H2O2). ABTS+, a soluble chromogen, can be monitored spectrophotometrically at 405 nm.

Platform

Absorbance microplate reader

Absorbance	405 nm
Recommended plate	Clear bottom

Components

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

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. 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

Trolox standard

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.

Images

Figure 1. Trolox dose-response was measured with the Amplite® Colorimetric Antioxidant Activity Assay Kit (Cat# 15900) in a 96-well clear plate using a SpectraMax microplate reader (Molecular Devices).