Solarbio Hydrogen Peroxide (H2O2) Kit Pengujian Konten

Komponen:

Reagen I: Acetone 100mL×1. Penyimpanan pada suhu 4℃. (Reagen yang disediakan sendiri)

Reagen Ⅱ: Bedak×1. Penyimpanan pada suhu 4℃. Solusi kerja: Add 3mL concentrated hydrochloric acid (37%) sebelum digunakan, fully dissolved. Unused reagents stored at 4 °C.

Reagen Ⅲ: 6mL×1. Penyimpanan pada suhu 4℃.

Reagen Ⅳ: 30mL×1. Penyimpanan pada suhu 4℃.

Standar: 1mL×1, 1mmol/mL H2O2 standard solution. Penyimpanan pada suhu 4℃.

Deskripsi Produk:

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. Di sisi lain, 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.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan.

Spektrofotometer/pembaca lempeng mikro, mesin sentrifugal meja, pipet yang dapat disesuaikan, kuvet kaca mikro/pelat dasar datar sumur 96, pipet transfer, acetone concentrated sulfuric acid (37% Hcl), mortar and ice.

Prosedur:

SAYA. Ekstraksi Sampel:

1. 1. Bacterial or cell sample: collect bacterial or cell sample to centrifuge, buang supernatannya; suggested 5 million with 1mL of regent I, splitting bacteria and cell with ultrasonication (kekuatan 20%, Waktu kerja 3s, interval 10 detik, untuk 30 waktu); centrifuge at 8000g and 4℃ for 10 menit, supernatant is placed on ice for test.
   2. Jaringan: take 0.1g tissue add 1 ml regent I, sepenuhnya digiling di atas es. Sentrifugasi di, 8000g and 4℃for 10 menit, supernatant is placed on ice for test.
   3. Serum: according to the proportion of per 100μL of serum (plasma) add 0.9mL regent I, campur dengan baik. Centrifuge at 8000g and 4℃ for 10 menit, supernatant is placed on ice for test.

II. Tekad prosedur:

• Pemanasan lebih cepat spektrofotometer/pembaca mikro untuk 30 menit, sesuaikan panjang gelombang ke 415 nm, atur nol dengan air suling.
• Incubate Solution Ⅱ, Ⅲ and Ⅳ at 37℃(mammals) atau 25℃ (other animals) water bath for more than 10 min minutes.
• Solusi kerja standar: 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
• Tambahkan reagen dengan daftar berikut (reaction in EP tube):

Add Regent Ⅳ to dissolve the precipitate (the step can remove the vegetable pigment with acetone for 3-5 waktu), and place it at room temperature for 5 menit, 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.

AKU AKU AKU. Perhitungan (For Microplate reader)

A. 96-Sehat plate

• Jumlah sel

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

• Berat sampel

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

• Konsentrasi protein

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

• Serum (plasma)volume

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

500: cell or bacteria amount, 104;

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

Vs: volume sampel, 0.25 ml; W: Berat sampel, G;

Ve: volume ekstraksi, 1 ml;

Cpr: Konsentrasi protein sampel, 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.

Catatan:

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.

Contoh eksperimental:

1. Mengambil 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. Mengambil 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.

Referensi：

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

Kimia Analisis, 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, dkk. 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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Spesifikasi teknis:

Limit of Detection ：0.0027 mol/mL

Rentang linier：0.0195-3 mol/mL