Total Anti-Oxidative Capability Assay Kit（A015-2）
First Edition (Revised on April, 2016)
[ INTENDED USE ]
The kit is a ABTS method for the in vitro quantitative measurement of total antioxidant capacity within serum, plasma, tissue homogenate, cells (or cell culture supernates).
[ REAGENTS AND MATERIALS PROVIDED ]
96-well strip plate
[ MATERIALS REQUIRED BUT NOT SUPPLIED ]
Plate readers with light filters of desired wavelength (405-425nm)
[ STORAGE OF THE KITS ]
1. Reagent 1: Buffer Solution. Can be stored at -20℃ for 6 months.
2. Reagent 2: ABTS Solution. Can be stored at -20℃ for 6 months. Avoid Illumination.
3. Reagent 3: H2O2 Stock Solution. Can be stored at -20℃ for 6 months.
4. Reagent 4: Peroxidase Stock Solution. Can be stored at -20℃ for 6 months.
5. Reagent 5: 10mM Trolox Solution. Can be stored at -20℃ for 6 months. Avoid Illumination.
[ REAGENT PREPARATION ]
1. Reagent 3 Solution Preparation: Dilute the stock solution with double distilled water (DDW) to 1000 times of its original volume.
2. ABTS Solution Preparation: Blend Reagent 1, Reagent 2 and Reagent 3 solution with the ratio of 76:5:4 in order to prepare the desired amount of ABTS solution. ABTS Solution should be preserved at RT without light and used within 30 min.
3. Reagent 4 Solution Preparation: Dilute the given reagent 4 with reagent 1 to the 10 times of its original volume. Reagent 4 solution should be prepared right before its usage with the amount needed.
[ SAMPLE PREPARATION ]
1. Aqueous Sample like Serum/Plasma
Pretreatment: Measure directly. Note, for plasma samples, EDTA is not a recommended coagulant.
2. Tissue Sample
Pretreatment: Weight the sample precisely and add the saline with ratio of 1g sample with 9ml saline. Homogenize the mixture in the ice water bath and the centrifuge the homogenate at 4℃, 12000 rpm/min for 5min. Extract the supernatant for further measurement.
Pretreatment: Collect no less than 1 million cells and add 200μl cold phosphate buffer solution. Disrupt the cells either by homogenization or sonication, and then centrifuge the homogenate at 4℃, 12000 rpm/min for 5min. Extract the supernatant for further measurement.
Note: For tissue sample and cells, calculation requires the protein concentration of the homogenate. It is recommended to use IS003 BCA Protein Quantification Kit to determine the protein concentration.
[ ASSAY PROCEDURE ]
Distilled Water (μl)
Trolox Solution (μl)
Reagent 4 Solution (μl)
ABTS Solution (μl)
|React at RT for 6 min. Read the optical density (OD) at 414nm with a plate reader.|
Note: It is recommended to dilute the Trolox solution given with distilled water to the concentration of 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM in order to draw the standard curve.
[ TEST PRINCIPLE ]
2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) can be oxidized to greenish ABTS+ in the presence of proper oxidants. The production of ABTS+ can be inhibited with antioxidants and thus the total antioxidant capacity can be calculated based on the optical density of ABTS+ at 414 or 734nm. Trolox is a water-soluble analog of vitamin E with the similar anti-oxidative capability and is applied as the antioxidant capacity equivalency.
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