Solarbio Superoksida Dismutase (MERUMPUT) Kit Uji Aktivitas

Superoxide Dismutase (MERUMPUT) Kit Uji Aktivitas

Catatan: Hal ini perlu untuk diprediksi 2-3 perbedaan sampel yang besar sebelum penentuan formal.

Peralatan Operasi: Spektrofotometer

Kucing No: BC0170

Ukuran: 50T/24S

Komponen:

Reagen ekstraksi: 60 mL×1. Penyimpanan pada suhu 4℃.

Reagent Ⅰ: 15 mL×1. Penyimpanan pada suhu 4℃.

Reagent Ⅱ: 160 μL×1. Penyimpanan pada suhu 4℃. Mix by pipetting after centrifugation.

Reagen Ⅲ: 11 mL×1. Penyimpanan pada suhu 4℃.

Reagent Ⅳ: Bedak×1. Penyimpanan pada suhu 4℃.

Reagen V: 2 mL×1. Penyimpanan pada suhu 4℃. Add Reagent Ⅳ to Reagent V before use and shake with an oscillator to mix thoroughly. It can be stored 3 bulan.

Deskripsi Produk:

Superoxide dismutase (MERUMPUT, EC 1.15.1.1) is widely found in animals, plants, microorganisms and cultured cells. It catalyzes the superoxide anion to form H2O2 and O2. SOD is not only the superoxide anion scavenging enzyme, but also the main H2O2 producing enzyme, which plays an important role in the biological antioxidant system.

Superoxide anion (O2-) is produced by the xanthine and xanthine oxidase reaction system. O2- can reduce blue tetrazole to create blue formazan, which has absorbance in 560 nm. SOD can remove O2- and inhibit the formation of methionine. The darker the blue color of the reaction solution, the lower the activity of SOD. The lighter the blue color of the reaction solution, the higher the activity of SOD.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:

Spektrofotometer, table centrifuge, pipet transfer, 1 mL kuvet kaca, mortir/homogenizer, es dan air suling.

Operation steps:

SAYA. Persiapan sampel:

1. Bakteri atau sel: collecting bacteria or cells into the centrifuge tube, discard supernatant after centrifugation. According to the proportion of bacteria or cells (104 cells): extraction solution volume (ml) of 1:5-10 to extract. It is suggested that 5 million of bacteria or cells amount with 1mL of Extraction reagent. Splitting the bacteria or cells with ultrasonication (ditempatkan di atas es, ultrasonic power 200W or 20%, working time 3s, interval 10s, ulangi untuk 30 waktu). Sentrifugasi di 8000 ×g untuk 10 menit pada suhu 4℃ untuk menghilangkan bahan yang tidak larut, dan mengambil supernatan di atas es sebelum pengujian.
2. Jaringan: according to the proportion of tissue weight (G): extraction solution volume (ml) of 1:5-10 to extract. It is suggested that 0.1 g of tissue with 1 mL of Extraction reagent and fully homogenized on ice.
3. Sentrifugasi di, 8000 ×g untuk 10 menit pada suhu 4℃ untuk menghilangkan bahan yang tidak larut, dan mengambil supernatan di atas es sebelum pengujian.
4. Serum (plasma) sample: detect sample directly.

II. Tekad prosedur:

1. Preheat the spectrophotometer for 30 menit, sesuaikan panjang gelombang ke 560 nm and set zero with distilled water.
2. Keep Reagent Ⅰ, Reagen Ⅲ, Reagent V in water bath for more than 5 minutes at 37℃(mammal) or

25℃ (other species).

3. Tambahkan reagen dengan daftar berikut:

Mix thoroughly and the mixture is incubated at room temperature for 30 menit. Add the mixture into 1mL glass cuvette, and detect the absorbance value of each tube at 560 nm. ΔAT=AT-AC，ΔAB=AB1-AB2. If there is precipitation at the bottom, mix thoroughly and then measure.

AKU AKU AKU. Perhitungan:

1. Inhibition percentage:

Inhibition percentage=[ΔAB -ΔAT]÷ΔAB× 100%

The inhibition percentage should be in 30%~70% (the value close to 50% will have a more accurate result). If the calculated inhibition percentage is less than 30% or more than 70%, it is usually necessary to adjust the sample addition amount and re determine. If the percentage of inhibition is too high, the sample should be diluted properly. If the percentage of inhibition is too low, the sample should be reprepared with a higher concentration.

2. Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the inhibition of 50% in the reaction system of the above xanthine
3. Perhitungan

A. Serum (plasma)sample

MERUMPUT (U/mL)=[P÷(1-P)×Vrv]÷Vs×F=11.4×P÷(1-P)×F

B. Jaringan, bakteri atau sel yang dikultur

A) Konsentrasi protein:

MERUMPUT (U/mL prot) = [P÷(1-P)×Vrv]−(Vs×Cpr)×F=11.4×P÷(1-P)÷Cpr×F

B) Berat sampel

MERUMPUT (berat U/g) = [P÷(1-P)×Vrv]−(W×Vs^Vsv)×F=11.4×P÷(1-P)÷W×F

C) Bacteria or cell amount

MERUMPUT (U/104 cell)=[P÷(1-P)×Vrv]−(500×Vs^Vsv)×F=0.0228×P÷(1-P)×F

Tali: Volume reaksi total, 1.026 ml; Vs: Volume sampel, 0.09 ml;

Vsv: Volume ekstraksi, 1 ml;

Cpr: Konsentrasi protein sampel, mg/mL; W: Berat sampel, G;

500: Jumlah total bakteri dan sel, 5 juta. P: Inhibition percentage, %;

F: Sample dilution multiple.

Catatan:

1. The Sample and Reagent Ⅱ should be placed on ice when
2. When there are many samples, the working solution (including Reagent I, II and III) can be configured according to the table. Reagent V must be added
3. After the reaction completed, there may be precipitation formed, which can be determined after

Experimental Examples:

1. 1 g of Echinochloa crusgalli is added into 1 mL of Extraction reagent for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT = AT – AC = 0.335-0.012 = 0.323, ΔAB = AB1 – AB2 = 0.957-0.003 = 0.954. Inhibition percentage = (ΔAB- ΔAT)÷ ΔAB × 100% = 72%, and the enzyme activity is calculated according to the sample mass.

SOD activity (massa U/g) = 11.4 × Inhibition percentage (1-Inhibition percentage) × W = 293.14 massa U/g.

2. 1 mL of Extraction reagent is added to 0.1 g of rat spleen for homogenization. After the supernatant is taken, the operation is carried out according to the determination steps. The results showed that ΔAT= AT- AC = 0.563-0.213 = 0.35, ΔAB= AB1- AB2= 0.957-0.003 = 0.954, inhibition percentage = (ΔAB -ΔAT)÷ ΔAB×100% =31%

SOD activity (massa U/g) = 11.4 × Inhibition percentage (1-Inhibition percentage) ×W = 196.71 massa U/g.

3. 10 million cells is extracted and centrifuged by adding 1 mL reagen Ekstraksi, and then the operation is performed according to the determination steps. The results are as follows: ΔAT =AT – AC=614-0.015 = 0.599, ΔAB= AB1- AB2= 0.944-0.005 = 0.939, inhibition percentage = (ΔAB- ΔAT) ×ΔAB× 100% = 36.21%

SOD activity (U/104 cell) = Inhibition percentage ÷ (1-Inhibition percentage)× VTS]−(1000× VS ÷ VTS) = 0.0065 U/104 cell.

Referensi:

• Spitz DR, Oberley L W. An assay for superoxide dismutase activity in mammalian tissue homogenates[J]. Biokimia Analitik, 1989,179(1):8-18.
• Masayasu M, Hiroshi Y. Metode pengujian aktivitas superoksida dismutase yang disederhanakan untuk penggunaan klinis[J]. Clinica Chimica Acta, 1979,92(3):337-342.

Produk-produk terkait:

BC0190/BC0195 Polyphenol Oxidase (PPO) Kit Uji Aktivitas

BC0210/BC0215 Phenylalnine Ammonialyase (PAL) Kit Uji Aktivitas

BC0200/BC0205 Catalase (CAT) Kit Uji Aktivitas

BC0090/BC0095 Peroxidase (POLONG) Kit Uji Aktivitas