Solarbio Capacità antiossidante totale (T-AOC) Kit di analisi

Capacità antiossidante totale (T-AOC) Kit di analisi

Nota: Prendi due o tre campioni diversi per la previsione prima del test.

Attrezzatura operativa: Spettrofotometro

Gatto n: BC1310

Misurare：50T/48S

Componenti:

Estrarre la soluzione: Liquido 50 ml×1. Conservazione a 4 ℃, precool before use.

Reagente I: Liquido 35 ml×1. Conservazione a 4 ℃.

Reagente II: Liquido 20 ml×1. Storage at 4℃ in shadow.

Reagente III: Liquido 5 ml×1. Storage at 4℃ in shadow.

Standard: Polvere×1, 10 mg of FeSO4·7H2O. Soluzione funzionante: aggiungere 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): Reagente I : Reagente II: Reagent III= 7:1:1, incubate at 37℃ before use.

Descrizione del prodotto:

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.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, bagnomaria a temperatura costante, low temperature centrifuge, 1 mL glass cuvette and distilled water.

Procedura:

IO. preparazione del campione:

1. Siero, plasma, saliva, or urine samples

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

2. Cells or tissue campione

Prendere 1-2 million cells or 0.1 g of tissue, aggiungere 1.0 ml di soluzione di estratto. Use homogenate or ultrasound to fully break up cells and release antioxidant, centrifugare a 10000 r/min and 4℃ for 5 min, take supernatant for test. Measure the concentration of protein if needed.

II. Determination procedure:

1. Dilute 40 μmol/mL FeSO4 standard solution to 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125 µmol/ml, Prendere 500 μL of standard solution (distilled water for blank control), add to 500 μL di Reagente II. Mix thoroughly for 10 min, detect the absorbance in 593 nm, calculate ΔA=AS-AB. (COME: 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, regolare la lunghezza d'onda su 593 nm and set zero with distilled
3. Aggiungere i reagenti con il seguente elenco:

III. Calcolo:

1. Crea curva standard

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. Definizione di unità: the sample antioxidant capacity is indicated by the standard liquid ion concentration required for the same absorbance change(ΔA).

UN. Protein concentration:

Capacità antiossidante totale (μmol/mg prot) = x × Vrv÷ (Vs× Cpr) = 34× x ÷ Cpr

B. Campione weight

Capacità antiossidante totale (μmol/g weight) = x × Vrv÷ (Vs ÷ Vsv ×W) =34× x÷ W

C. Cell amount

Capacità antiossidante totale (μmol/104cell) = x × Vrv÷ (Vs ×Vsv÷n) = 34× x÷ n

D. Solution volume

Capacità antiossidante totale (µmol/ml) =x× Vrv÷ Vs =34×x

Corda: volume totale di reazione, 1.02 ml; Contro: sample volume, 0.03 ml;

Vsv: extraction volume, 1 ml; W: sample weight, G;

Cpr: concentrazione delle proteine ​​del campione, mg/ml;

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

Nota:

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)-UN(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.

Riferimenti：

[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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