Peroksidase (POLONG) Kit Uji Aktivitas

Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.

Peralatan Operasi: Spektrofotometer Nomor Katalog: BC0090 Sizes：50T/48S

Komponen:

Extract solution: 60 mL×1. Penyimpanan pada suhu 4℃.

Reagen I: 40 mL×1. Penyimpanan pada suhu 4℃.

Reagen II: 0.5 mL ×1. Penyimpanan pada suhu 4℃. Centrifuge before use. Mengambil 0.22 mL of reagent II and add 3.33 mL of reagent I, mix it for later use (about 27T). Prepare it for immediate use, or it can be prepared in proportion according to the sample volume.

Reagen III: 10 mL×1. Penyimpanan pada suhu 4℃.

Deskripsi Produk

Peroksidase (POLONG, EC 1.11.1.7) widely exists in animals, plants, and microorganisms. It can catalyze the oxidation of phenols and amines by hydrogen peroxide, and has the dual effect of eliminating toxicity of hydrogen peroxide, phenols, and amines. In the presence of hydrogen peroxide, POD can catalyzes H2O2 oxidize specific substrates to produce one substance which has a absorption at 470 nm.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan

Spektrofotometer, mesin sentrifugal meja, transferpettor, 1 mL kuvet kaca, mortir/homogenizer, es dan air suling.

Prosedur

SAYA. Persiapan sampel:

A. Bakteri atau sel

Collecting bacteria or cells into the centrifuge tube, the supernatant is discarded after centrifugation. It is suggested to take about 5 million bacteria/cell and add 1 mL of Extract solution. Bacteria or cell is splitted by ultrasonication (Power: 20% or 200 W, work time 3s, interval 10s, ulangi untuk 30 waktu). Sentrifugasi di 8000 rpm and 4℃ for 10 menit, the supernatant is used for test.

B. Jaringan

It is recommended to take about 0.1 g of tissue and add 1 mL of Extract solution, fully grinding on ice.

Sentrifugasi di 8000 rpm untuk 10 minutes at 4℃, the supernatant is used for test.

C. Serum (plasma) sample: Detect sample

II. Tekad prosedur

1. Preheat spectrophotometer for 30 menit, sesuaikan panjang gelombang ke 470 nm, atur nol dengan sulingan
2. Place Reagent I, Reagent II and Reagent III at 37℃ (mammal) or 25℃(other species) untuk 10 minutes before
3. Tambahkan reagen dengan daftar berikut:

The above reagents are added into 1 mL glass cuvette in sequence, immediately mixed and timed. The absorbance values A1 for 30 s and A2 for 90s at 470 nm are recorded, ΔA＝A2-A1.

AKU AKU AKU. Perhitungan

SAYA. Serum (plasma)sample

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the absorbance of 0.01 change at 470 nm in the reaction system per minute every milliliter of serum (plasma).

POLONG(U/mL)=ΔA×Vrv÷Vsv÷0.01÷T =7133×ΔA

II. Jaringan, bacteria or culture cells

A. Konsentrasi protein

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the absorbance of 0.01 change at 470 nm in the reaction system per minute every milligram protein.

POLONG(U/mg prot)＝ΔA×Vrv÷(Vsv×Cpr)÷0.01÷T =7133×ΔA÷Cpr

B. Berat sampel

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the absorbance of 0.01 change at 470 nm in the reaction system per minute every gram tissue.

POLONG(U/g fresh weight)＝ΔA×Vrv÷(W× Vsv÷Vs)÷0.01÷T =7133×ΔA÷W

C. jumlah sel

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the absorbance of 0.01 change at 470 nm in the reaction system per minute every 10 thousand bacteria or cells.

POLONG(U/104 cell)＝ΔA×Vrv÷(500×Vsv÷Vs)÷0.01÷T =14.27×ΔA

Tali: Volume reaksi total, 1.07 ml; Vsv: Total supernatant volume, 0.015 ml; Vs: Extract solution volume, 1 ml;

T: Reaction time, 1 menit;

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

500: Total number of bacteria or cells, 5 juta.

Catatan:

1. If there are many samples to be determined at one time, the mixture of Reagent I, II, III and distilled water can be prepared in proportion, and the mixture can be placed at 37℃ (mammalian) or 25℃ (other species) for more than 10 menit. 15 μL sampel dan 1055 μL of mixture can be added for determination.
2. If ΔA is less than 0.005, the reaction time can be extended to 5 menit. If ΔA is greater than 0.5 or there are more bubbles in the reaction solution, the sample can be diluted with the extract and determined, and the calculation formula is multiplied by the corresponding dilution.
   

   Referensi

   • Reuveni R . Peroxidase Activity as a Biochemical Marker for Resistance of Muskmelon ( Cucumis melo ) to Pseudoperonospora cubensis[J]. Phytopathology, 1992,82(7).
   • Doerge D R , Divi R L , Churchwell M I . Identification of the Colored Guaiacol Oxidation Product Produced by Peroxidases[J]. Biokimia Analitik, 1997,250(1):10-17.

   Produk-produk terkait

   BC0190/BC0195 Polyphenol Oxidase (PPO) Activity Assay Kit BC0210/BC0215 Phenylalanine Ammonia Lyase (PAL) Activity Assay Kit BC0170/BC0175 Superoxide Dismutase (MERUMPUT) Activity Assay Kit BC0200/BC0205 Catalase (CAT) Kit Uji Aktivitas