Solarbio ukupni antioksidacijski kapacitet (T-AOC) Komplet za analizu

Ukupni antioksidacijski kapacitet (T-AOC) Komplet za analizu

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar

Mačka ne: BC1310

Veličina：50T/48s

Komponente:

Otopina ekstrakta: Liquid 50 ml × 1. Skladištenje na 4 ℃, precool before use.

Reagens i: Liquid 35 ml × 1. Skladištenje na 4 ℃.

Reagens II: Liquid 20 ml × 1. Storage at 4℃ in shadow.

Reagens iii: Liquid 5 ml × 1. Storage at 4℃ in shadow.

Standard: Prah × 1, 10 mg of FeSO4·7H2O. Radno rješenje: dodati 0.9 mL of distilled water and 20μL of concentrated sulfuric acid (H2SO4) to forms 40 µmol/mL FeSO4 standard solution. Solution mixture (prepare when the solution will be used): Reagens i : Reagens II: Reagent III= 7:1:1, incubate at 37℃ before use.

Opis proizvoda:

This kit is used to detect the total antioxidant levels of antioxidants and antioxidant enzymes in the samples. It is mainly used in the study of biological, medical and pharmaceutical studies to detect the total antioxidant capacity of antioxidant solutions.

In acid environment, Fe3+ -TPTZ are reduced to blue Fe2+ -TPTZ. The color reaction reflects the total antioxidant capacity.

Potrebni reagensi i oprema, ali nisu pruženi:

Spektrofotometar, Stalna temperaturna vodena kupka, low temperature centrifuge, 1 mL glass cuvette and distilled water.

Postupak:

Ja. Priprema uzorka:

1. Serum, plazma, saliva, or urine samples

Plazma (anticoagulation with heparin or sodium citrate, avoid using EDTA), centrifuga na 5000 rpm/min for 10 min, take supernatant for test. Take serum, saliva or urine samples for direct determination. Also, you can store at -80℃ and detect within 30 dana.

2. Cells or tissue uzorak

uzeti 1-2 million cells or 0.1 g tkiva, dodati 1.0 ML otopine ekstrakta. Use homogenate or ultrasound to fully break up cells and release antioxidant, centrifuga na 10000 r/min and 4℃ for 5 min, take supernatant for test. Measure the concentration of protein if needed.

Ii. Determination procedure:

1. Razrijediti 40 μmol/mL FeSO4 standard solution to 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 μmol/ml, take 500 μL of standard solution (distilled water for blank control), add to 500 μL of Reagent II. Mix thoroughly for 10 min, detect the absorbance in 593 NM, calculate ΔA=AS-AB. (KAO: standard solution tube, Ab: blank control tube.) The final concentration of Fe2+ is 0.05、0.025、0.0125、0.00625、

0.003125、0.00156 μmol/ml.

2. Preheat the spectrophotometer 30 min, prilagoditi valnu duljinu na 593 nm and set zero with distilled
3. Dodajte reagense sa sljedećim popisom:

Ime reagensa	Prazna cijev (Ab)	Epruveta (NA)
Solution mixture (μL)	900	900
Uzorak (μL)		30
Double distilled water (μL)	120	90
Mix thoroughly and react for 10 min, Postavite nulu s destiliranom vodom, detect the absorbance in 593 NM. Calculate ΔA’=AT – Ab. (Bilješka: The blank tube just needs to be tested once or twice in every experiment)

Iii. Izračunavanje:

1. Create standard curve

Take the Fe2+ final concentration as X-axis, △A as Y-axis, create standard curve, get linear regression equation y=kx+ b, take ΔA’ into the equation to get x (μmol/ml).

2. Definicija jedinice: the sample antioxidant capacity is indicated by the standard liquid ion concentration required for the same absorbance change(ΔA).

A. Protein concentration:

Ukupni antioksidacijski kapacitet (μmol/mg prot) = x × Vrv÷ (Vs× Cpr) = 34× x ÷ Cpr

B. Uzorak weight

Ukupni antioksidacijski kapacitet (μmol/g weight) = x × Vrv÷ (Vs ÷ Vsv ×W) =34× x÷ W

C. Cell amount

Ukupni antioksidacijski kapacitet (μmol/104cell) = x × Vrv÷ (Vs ×Vsv÷n) = 34× x÷ n

D. Solution volume

Ukupni antioksidacijski kapacitet (μmol/ml) =x× Vrv÷ Vs =34×x

Vrpca: Ukupni volumen reakcije, 1.02 ml; Vs: volumen uzorka, 0.03 ml;

VSV: extraction volume, 1 ml; W: Težina uzorka, g;

CPR: Koncentracija proteina uzorka, mg/ml;

n: cell amount, unit based on 104 (ten thousand).

Bilješka:

1. Reagent II is irritated to human body, please wear lab clothes and latex
2. The samples should not be appeared blue under acidic condition, or it will interference sample result of the
3. Detergent such as Tween, Triton, NP-40 and reductants such as DTT, mercapto ethanol should not be added in the
4. If the absorbance value determined by the sample is beyond the standard curve range, the sample should be diluted or concentrated properly before
5. The kit should be store at2-8℃.

Examples:

1. Add 0.1g shamrock to 1mL extract solution and grind thoroughly on ice, take supernatant, follow the determination procedure to operate, with 96-well flat-bottom plates to calculate: ΔA=A(T)-A(B)=0.909-0.148=0.761, standard curve: y=21.056x-0.0087, calculate x=0.037, according to mass of sample to calculate Total antioxidant capacity (μmol/g mass) =34×x÷W=34×0.037÷0.1=12.85 μmol/g mass.

Reference：

[1] Pellegrini N, Serafini M, Salvatore S, et al. Total antioxidant capacity of spices, dried fruits, nuts,

pulses, cereals, and sweets consumed in Italy assessed by three different in vitro assays[J]. Molecular nutrition & food research, 2006, 50(11): 1030-1038.

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