Total Anti-Oxidative Capability Assay Kit (A015-1)

Instruction manual

First Edition (Revised on April, 2016)

[ INTENDED USE ]

The kit is a colorimetric method for the in vitro quantitative measurement of total antioxidant capacity(T-AOC) in animal blood serum, tissues, plant extracts, etc..

There are close correlations between T-AOC of organism defense system and health level. This defense system includes 2 systems: enzymatic & nonenzymatic. In enzymatic system, microelements act as active centers for various enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), glutathione S-transferase (GST), etc. Nonenzymatic system mainly include vitamins, amino acids & metalloproteins such as V_E, carotene, Vc, cysteine, methionine, tryptophane, histidine, glucose, copper-protein, transferrin, lactoferrin, etc. There are 3 main pathways of antioxidation in defense system: (1) Eliminate free radicals and active oxygen in order to avoid lipid peroxidation; (2) Decompose peroxides to interrupt peroxidation chain; (3) Remove catalytic metal ions. There are synergetic effect, compensation & dependency between different components of defense system.

 

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents	Quantity(96T/48T)	Reagents	Quantity(96T/48T)
Reagent  1	2×60ml/1×60ml	Reagent  4	1×24ml/1×12ml
Reagent  2	2/1	Reagent  5	1×24ml/1×12ml
Reagent  3	1×5ml/1×3ml	Instruction manual	1/1
Reagent  3 Diluent	1×60ml/1×60ml

Note: Reagent 5 freezes in cold days, when use, please place it in 37℃ water bath until it becomes limpid completely.

 

[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1.  A spectrophotometer capable of measuring absorbance at 520nm

2. Thermostatic water bath or air bath capable of controlling temperature at 37℃

3. Desk centrifuge

4. Micropipets and tips

5. Vortex mixer

6. A source of pure water (preferably double distilled water and double distilled water)

 

[ STORAGE OF THE KITS ]

This kit can be stored at 2~8℃ hermetically for 6 months.

Reagent 1: Colorless liquid, can be stored at 2~8℃;

Reagent 2: White crystals, can be stored at 2~8℃;

Reagent 3: Yellow stock solution, can be stocked at 2~8℃ away from light;

Reagent 3 diluent: Can be stored at 2~8℃;

Reagent 4: Colorless thick liquid, can be stored at room temperature;

Reagent 5: Colorless liquid, can be stored at room temperature.

 

[ REAGENT PREPARATION ]

1.  Reagent 2 Preparation: Add 120ml distilled water in each vial, dissolve completely (this powder is difficult to dissolve, if you want to increase dissolving rate, please place it in 37℃ water bath.

2.  Reagent 3 working solution preparation: Dilute Reagent 3 with Reagent 3 diluent at ratio of 1:19, please use this working solution soon after preparation.

 

[ SAMPLE PREPARATION ]

1.  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 for 5min. Extract the supernatant for further measurement.

Note: For tissue samples, calculation requires the protein concentration of the homogenate. It is recommended to use IS003 BCA Protein Quantification Kit to determine the protein concentration.

2.  Whole blood

Collect whole blood using heparin as an anticoagulant. Assay immediately, take 0.02 ml fresh blood, add 0.18 ml double distilled water, mix sufficiently to prepare 1:9 diluted whole blood. Or stored the whole blood at -80℃ for future use.

Note: EDTA is not a recommended coagulant.

[ ASSAY PROCEDURE ]

1. Blood serum/plasma T-AOC assay

Operation table:

	Sample tube (U) (ml)	Contrast tube (C) (ml)
Reagent 1	1	1
Serum/plasma	a*
Reagent 2	2	2
Reagent 3 working solution	0.5	0.5
Mix sufficiently by vortex, place in 37℃ water bath for 30 minutes
Reagent 4	0.1	0.1
Serum/plasma		a*
Mix sufficiently, place for 10 minutes, transfer in cuvettes of 25px light path, measure OD values of all tubes at 520nm (adjust zero by distilled water).

* Referenced sampling volume: For blood serum/plasma, a*=0.1ml.

 

2. Tissue T-AOC assay

Operation table:

	Sample tube (U) (ml)	Contrast tube (C) (ml)
Reagent 1	1	1
10% tissue homogenate	a*
Reagent 2	2	2
Reagent 3 working solution	0.5	0.5
Mix sufficiently by vortex, place in 37℃ water bath for 30 minutes
Reagent 4	0.2	0.2
10% tissue homogenate		a*
Reagent 5	0.2	0.2
Mix sufficiently, place for 10 minutes, transfer in cuvettes of 25px light path, measure OD values of all tubes at 520nm (adjust zero by distilled water).

* Referenced sampling volume: For 10% tissue homogenate, a*=0.1~0.2ml.

 

3. Whole blood T-AOC assay

Operation table:

	Sample tube (U) (ml)	Contrast tube (C) (ml)
Reagent 1	1	1
1:9 diluted whole blood	a*
Reagent 2	2	2
Reagent 3 working solution	0.5	0.5
Mix sufficiently by vortex, place in 37℃ water bath for 30 minutes
Reagent 4	0.1	0.1
1:9 diluted whole blood		a*
Mix sufficiently, place for 10 minutes, transfer in cuvettes of 25px light path, measure OD values of all tubes at 520nm (adjust zero by distilled water).

* Referenced sampling volume: For 1:9 diluted whole blood, a*=0.05ml.

 

[ IMPORTANT NOTE ]

1.  If you don't want do T-AOC assay immediately, then please store samples below -20℃, Colder preservation leads to longer guarantee period.

2.  Add samples and reagents vertically, never drop on tube surface.

3.  Sample can be added after Reagent 1, but please mix sufficiently.

4.  If you use microscale samples such as eardrum tissue, dental pulp, etc, then please cleanse test tubes with boiled soapsuds completely.

5.  Please separate test tubes to assay T-AOC, total amino acids, Vc, Ve away from test tube to assay SOD, GSH-PX, ATPase, etc, in order to avoid affecting enzyme activities.

6.  When you wash test tubes to assay T-AOC, total amino acids, Vc, VE, please wash them by boiled hot detergent solution at first, then rinse them by tap water more than 10 times, rinse by distilled water once, oven drying or open-air drying. These procedures are in order to avoid affecting other enzymes’ measuring results.

7.   During procedures, some sediments appear in test tubes after 30 minutes in 37℃ water bath, these sediments disappear after adding Reagent 4, they won't affect results.

8.   lt is better to mix by vortex in order to make liquid mixed welll from top tobottom.

9.   Reagent 4 is hard to suck and blow, when use transfer R4, please suck and blow it slowly.

10.  This kit can be used in scientific research and laboratory only.

 

[ TEST PRINCIPLE ]

Organism contains various antioxidative compounds which can reduce Fe3+ to Fe2+, Fe2+ can react with phenanthrolin to produce stable complex. It is able to calculate T-AOC by measuring OD values.

[ DOWNLOAD ]

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