Solarbio Hydrogen Peroxide (H2O2) Kit de ensayo de contenido

Atributo	Detalles
Nota	Tome dos o tres muestras diferentes para predecir antes de la prueba.
Instrumento de detección	espectrofotómetro / Microplate reader
No gato	BC3595
Tamaño	100T/96S

Componentes:

reactivo yo: Acetone 100mL×1. Almacenamiento a 4 ℃. (Self-provided reagent)

Reactivo ⅱ: Polvo × 1. Almacenamiento a 4 ℃. Solución de trabajo: Add 3mL concentrated hydrochloric acid (37%) antes de usar, fully dissolved. Unused reagents stored at 4 °C.

Reactivo Ⅲ: 6mL×1. Almacenamiento a 4 ℃.

Reactivo ⅳ: 30mL×1. Almacenamiento a 4 ℃.

Estándar: 1mL×1, 1mmol/mL H2O2 standard solution. Almacenamiento a 4 ℃.

Descripción del Producto:

H2O2 is the most common reactive oxygen molecules in organisms. It is mainly produced by the catalyzation of SOD and XOD and degraded by the catalyzation of CAT and POD. H2O2, which is not only one of the important reactive oxygen, but also the hub of mutual conversion of reactive oxygen. Por un lado, H2O2 can directly or indirectly oxidize intracellular nucleic acids, proteins and other biological macromolecules, and damage cell membranes, thus accelerating the aging and disintegration of cells. Por otro lado, H2O2 is also a key regulatory factor in many oxidative emergency reactions.

H2O2 and titanium sulfate generate a yellow titanium peroxide complex with the characteristic absorption at 415 Nuevo Méjico.

Reactivos y equipos necesarios pero no suministrados.

Espectrofotómetro/lector de microplacas, centrífuga de escritorio, pipeta ajustable, cubeta de microvidrio/placa de fondo plano de 96 pocillos, pipeta de transferencia, acetone concentrated sulfuric acid (37% HCl), mortar and ice.

Procedimiento:

I. Extracción de muestras:

1. 1. Bacterial or cell sample: collect bacterial or cell sample to centrifuge, descartar el sobrenadante; suggested 5 million with 1mL of regent I, splitting bacteria and cell with ultrasonication (fuerza 20%, tiempo de trabajo 3s, intervalo 10s, para 30 veces); centrifuge at 8000g and 4℃ for 10 mín., supernatant is placed on ice for test.
   2. Tejido: take 0.1g tissue add 1 ml regent I, moliendo completamente sobre hielo. Centrifugar en, 8000g and 4℃for 10 mín., supernatant is placed on ice for test.
   3. Suero: according to the proportion of per 100μL of serum (plasma) add 0.9mL regent I, mezclar bien. Centrifuge at 8000g and 4℃ for 10 mín., supernatant is placed on ice for test.

II. Determinación procedimiento:

• Precaliente el espectrofotómetro/lector de microplacas para 30 mín., ajustar la longitud de onda a 415 Nuevo Méjico, poner a cero con agua destilada.
• Incubate Solution Ⅱ, Ⅲ and Ⅳ at 37℃(mamíferos) o 25 ℃ (other animals) water bath for more than 10 min minutes.
• Solución de trabajo estándar: If using a 96-well plate, dilute the 1mmol/mL standard solution to 2μmol/mL standard solution with acetone, and use a trace glass colorimetric method to dilute 1mmol/mL standard solution to 1μmol/mL standard solution
• Agregue reactivos con la siguiente lista (reaction in EP tube):

Reactivo (µL)	Tubo de ensayo (EN)	Tubo estándar (COMO)	Tubo de control (C.A.)
Muestra	250
Solución de trabajo estándar		250
Regente yo			250
Regent II	25	25	25
Regent III	50	50	50
4000gramo, room temperature centrifuge for 10 mins, descartar sobrenadante.
Regent IV	250	250	250

Add Regent Ⅳ to dissolve the precipitate (the step can remove the vegetable pigment with acetone for 3-5 veces), and place it at room temperature for 5 mín., Transfer 200 μL to a micro glass cuvette or 96-well plate and measure the absorbance at 415 Nuevo Méjico. The control tube need only be tested once or twice. Calculate ∆AT =AT-AC, ∆AS=AS-AC.

III. Cálculo (For Microplate reader)

A. 96-well lámina

• cantidad de celda

H2O2(μmol /104 celúla) = ∆AT÷(∆AS÷C)×Vs÷(500×Vs÷Ve)=0.004 × ∆AT ÷ ∆AS

• Peso de la muestra

H2O2(μmol/g)= ∆AT÷(∆AS÷C)×V1÷(Vs÷Ve×W)= 2×∆AT ÷ ∆AS ÷ W

• Concentración de proteínas

H2O2(μmol/mg de protección)= ∆AT ÷(∆AS÷C)×Vs÷(RCP×Vs)= 2×∆AT÷ ∆AS ÷ Cpr

• Suero (plasma)volumen

H2O2(µmol/mL)= ∆AT ÷(∆AS÷C)×10=20 × ∆AT÷ ∆AS

500: cell or bacteria amount, 104;

C: concentration of H2O2 standard solution, 2µmol/mL;

contra: volumen de la muestra, 0.25 ml; W.: Peso de la muestra, gramo;

ve: volumen de extracción, 1 ml;

RCP: concentración de proteína de la muestra, mg/mL;

10: serum dilution multiple. [0.1mL serum (plasma)+0.9mL regent I]÷0.1mL serum(plasma)=10.

B. micro glass cuvette:

Change the concentration of standard C-2μmol/mL in the above formula to C-1μmol/mL for calculation.

Nota:

1. As Solution, I is easily volatile, Solution I must be precooled before use. It must be ground on ice when grinding.
2. The solution in this kit is easily volatile. Please bring disposable gloves and masks.
3. If the absorbance value of the sample is greater than 1.1, it is recommended to dilute the sample with Reagent I before performing the measurement.

Ejemplos experimentales:

1. Llevar 0.1 g of heart and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceda de acuerdo con el procedimiento de determinación. After determination with 96 well flat-bottom plate, calculate ∆AT=AT-AC=0.083-0.046=0.039, ∆AS=AS-AC=0.824-0.046=0.778. The content is calculated according to the sample mass.

H2O2(μmol/g) =2×∆AT ÷ ∆AS ÷ W =1 μmol/g.

2. Llevar 0.1 g of tea and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceda de acuerdo con el procedimiento de determinación. After determination with 96 well flat-bottom plate, calculate ∆AT=AT-AC=0.258-0.003=0.255, ∆AS=AS-AC=0.637-0.003=0.634. The content is calculated according to the sample mass.

H2O2(μmol/g) =2×∆AT ÷ ∆AS ÷ W =4.5 μmol/g.

Referencias：

• Satterfield C N, Bonnell A interference in titanium sulfate method for hydrogen peroxide[j].

Química analítica, 1955, 27(7): 1174-1175.

• Amin V M, Olson N F. Spectrophotometric determination of hydrogen peroxide in milk[j]. Journal of Dairy Science, 1967, 50(4):461-464.
• Sima Y H, Yao J M, Hou Y S, et al. Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination[j]. Archives of insect biochemistry and physiology, 2011, 77(2): 72-80.

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Especificaciones técnicas:

Limit of Detection ：0.0027 µmol/mL

Rango lineal：0.0195-3 µmol/mL