Peroksidaza (Mahuna) Kit za ispitivanje aktivnosti

Bilješka: Uzmite dva ili tri različita uzorka za predviđanje prije testa.

Operativna oprema: Spektrofotometar Catalog Number: BC0090 Sizes：50T/48s

Komponente:

Otopina ekstrakta: 60 ml × 1. Skladištenje na 4 ℃.

Reagens i: 40 ml × 1. Skladištenje na 4 ℃.

Reagens II: 0.5 mL ×1. Skladištenje na 4 ℃. Centrifuge before use. uzeti 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.

Reagens iii: 10 ml × 1. Skladištenje na 4 ℃.

Opis proizvoda

Peroksidaza (Mahuna, EC 1.11.1.7) widely exists in animals, biljke, 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.

Potrebni reagensi i oprema, ali nisu pruženi

Spektrofotometar, stolna centrifuga, transferpettor, 1 ML staklena kiveta, minobacač/homogenizator, led i destilirana voda.

Postupak

Ja. Priprema uzorka:

A. Bakterije ili stanice

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 otopine ekstrakta. Bacteria or cell is splitted by ultrasonication (Power: 20% ili 200 W, Radno vrijeme 3s, Interval 10s, ponoviti za 30 vrijeme). Centrifuga na 8000 rpm and 4℃ for 10 minuta, the supernatant is used for test.

B. Tkivo

It is recommended to take about 0.1 g of tissue and add 1 ML otopine ekstrakta, fully grinding on ice.

Centrifuga na 8000 RPM za 10 minutes at 4℃, the supernatant is used for test.

C. Serum (plazma) uzorak: Detect sample

Ii. Odlučnost postupak

1. Predgrijan spektrofotometar za 30 minuta, prilagoditi valnu duljinu na 470 NM, set zero with distilled
2. Place Reagent I, Reagent II and Reagent III at 37℃ (sisavac) ili 25 ℃(Ostale vrste) za 10 minutes before
3. Dodajte reagense sa sljedećim popisom:

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.

Iii. Calculations

Ja. Serum (plazma)uzorak

Definicija jedinice: 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 (plazma).

Mahuna(U/ml)=ΔA×Vrv÷Vsv÷0.01÷T =7133×ΔA

Ii. Tkivo, bacteria or culture cells

A. Koncentracija proteina

Definicija jedinice: 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.

Mahuna(U/Mg Prot)＝ΔA×Vrv÷(Vsv×Cpr)÷0.01÷T =7133×ΔA÷Cpr

B. Težina uzorka

Definicija jedinice: 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.

Mahuna(U/g svježa težina)＝ΔA×Vrv÷(W× Vsv÷Vs)÷0.01÷T =7133×ΔA÷W

C. Cellamount

Definicija jedinice: 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.

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

Vrpca: Ukupni volumen reakcije, 1.07 ml; VSV: Total supernatant volume, 0.015 ml; Vs: Extract solution volume, 1 ml;

T: Vrijeme reakcije, 1 minuta;

CPR: Koncentracija proteina uzorka, mg/ml; W: Težina uzorka, g;

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

Bilješka:

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) ili 25 ℃ (Ostale vrste) for more than 10 minuta. 15 μL of sample and 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 minuta. 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.
   

   Reference

   • 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]. Analitička biokemija, 1997,250(1):10-17.

   Povezani proizvodi

   BC0190/BC0195 polifenol oksidaza (PPO) Activity Assay Kit BC0210/BC0215 Phenylalanine Ammonia Lyase (Šal) Activity Assay Kit BC0170/BC0175 Superoxide Dismutase (TRAVNJAK) Activity Assay Kit BC0200/BC0205 Catalase (MAČKA) Kit za ispitivanje aktivnosti