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Total Anti-Oxidative Capability Assay Kit（A015-2）

Instruction manual

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 ]

Reagents

Quantity(96T)

Reagents

Quantity(96T)

Reagent 1

1×20ml

Reagent 4

1×0.2ml

Reagent 2

1×1ml

Reagent 5

1×0.1ml

Reagent 3‍

1×0.5ml

96-well strip plate

1
Instruction manual 1


[ 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.

3.  Cells

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 ]

Operation table:


Blank  

Standard 

Sample

Distilled Water (μl)

10

Trolox Solution (μl)


10

Sample (μl)



10

Reagent 4 Solution (μl)

202020

ABTS Solution (μl)

170170170
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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