Solarbio Hydrogen Peroxide (H2O2) Komplet za ispitivanje sadržaja

Atribut	pojedinosti
Bilješka	Uzmite dva ili tri različita uzorka za predviđanje prije testa
Instrument za otkrivanje	Spektrofotometar / Microplate reader
Mačka ne	BC3595
Veličina	100T/96S

Komponente:

Reagens i: Acetone 100mL×1. Skladištenje na 4 ℃. (Self-provided reagent)

Reagens ⅱ: Prah × 1. Skladištenje na 4 ℃. Radno rješenje: Add 3mL concentrated hydrochloric acid (37%) Prije upotrebe, fully dissolved. Unused reagents stored at 4 °C.

Reagens ⅲ: 6ml × 1. Skladištenje na 4 ℃.

Reagens ⅳ: 30ml × 1. Skladištenje na 4 ℃.

Standard: 1ml × 1, 1mmol/mL H2O2 standard solution. Skladištenje na 4 ℃.

Opis proizvoda:

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. On the one hand, 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. On the other hand, 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 NM.

Potrebni reagensi i oprema, ali nisu pruženi.

Spectrophotometer/microplate reader, stolna centrifuga, Podesiva pipeta, micro glass cuvette/96 well flat-bottom plate, PRIJENOS, acetone concentrated sulfuric acid (37% HCl), mortar and ice.

Postupak:

Ja. Sample Extraction:

1. 1. Bacterial or cell sample: collect bacterial or cell sample to centrifuge, discard the supernatant; suggested 5 million with 1mL of regent I, splitting bacteria and cell with ultrasonication (vlast 20%, Radno vrijeme 3s, Interval 10s, za 30 vrijeme); centrifuge at 8000g and 4℃ for 10 min, supernatant is placed on ice for test.
   2. Tkivo: take 0.1g tissue add 1 ml regent I, fully grinding on ice. Centrifuga na, 8000g and 4℃for 10 min, supernatant is placed on ice for test.
   3. Serum: according to the proportion of per 100μL of serum (plazma) add 0.9mL regent I, Dobro promiješajte. Centrifuge at 8000g and 4℃ for 10 min, supernatant is placed on ice for test.

Ii. Odlučnost postupak:

• Preheat spectrophotometer/microplate reader for 30 min, prilagoditi valnu duljinu na 415 NM, Postavite nulu s destiliranom vodom.
• Incubate Solution Ⅱ, Ⅲ and Ⅳ at 37℃(mammals) ili 25 ℃ (other animals) water bath for more than 10 min minutes.
• Standard working solution: 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
• Dodajte reagense sa sljedećim popisom (reaction in EP tube):

Reagens (μL)	Test Tube (NA)	Standard Tube (KAO)	Control Tube (Ac)
Uzorak	250
Standard working solution		250
Regent I			250
Regent II	25	25	25
Regent III	50	50	50
4000g, room temperature centrifuge for 10 mins, discard supernatant.
Regent IV	250	250	250

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

Iii. Izračunavanje (For Microplate reader)

A. 96-well plate

• Količina ćelije

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

• Težina uzorka

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

• Koncentracija proteina

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

• Serum (plazma)volume

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

500: cell or bacteria amount, 104;

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

Vs: volumen uzorka, 0.25 ml; W: Težina uzorka, g;

VE: extraction volume, 1 ml;

CPR: Koncentracija proteina uzorka, mg/ml;

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

B. micro glass cuvette:

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

Bilješka:

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.

Experimental examples:

1. uzeti 0.1 g of heart and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceed according to the determination procedure. 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. uzeti 0.1 g of tea and add 1 mL of Reagent I for sample processing. After centrifugation to take all the supernatant, proceed according to the determination procedure. 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.

Reference：

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

Analytical Chemistry, 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.

Povezani proizvodi:

BC0020/BC0025 Malondialdehyde(MDA) Komplet za ispitivanje sadržaja

BC1090/BC1095 Xanthine Oxidase(XOD) Kit za ispitivanje aktivnosti

BC0690/BC0695 Glucose Oxidase(GOD) Kit za ispitivanje aktivnosti

Technical Specifications:

Limit of Detection ：0.0027 μmol/ml

Linear Range：0.0195-3 μmol/ml