Peroxidase (POD) Aktivitätstest-Kit

Notiz: Nehmen Sie vor dem Test zwei oder drei verschiedene Proben zur Vorhersage.

Betriebsausrüstung: Spektrophotometer Katalognummer: BC0090 Sizes：50T/48S

Komponenten:

Lösung extrahieren: 60 ml×1. Lagerung bei 4℃.

Reagenz I: 40 ml×1. Lagerung bei 4℃.

Reagenz II: 0.5 mL ×1. Lagerung bei 4℃. Centrifuge before use. Nehmen 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.

Reagenz III: 10 ml×1. Lagerung bei 4℃.

Produktbeschreibung

Peroxidase (POD, EC 1.11.1.7) widely exists in animals, Pflanzen, und Mikroorganismen. 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.

Erforderliche, aber nicht bereitgestellte Reagenzien und Ausrüstung

Spektrophotometer, Tischzentrifuge, transferpettor, 1 ML Glass Cuvette, Mörtel/Homogenisator, Eis und destilliertes Wasser.

Verfahren

ICH. Probenvorbereitung:

A. Bakterien oder Zellen

Sammeln von Bakterien oder Zellen in die Zentrifugenröhre, the supernatant is discarded after centrifugation. It is suggested to take about 5 Millionen Bakterien/Zellen und hinzufügen 1 ml Extraktlösung. Bacteria or cell is splitted by ultrasonication (Leistung: 20% oder 200 W, Arbeitszeit 3s, Intervall 10s, Wiederholen Sie 30 mal). Zentrifuge bei 8000 Drehzahl und 4 ℃ für 10 Protokoll, the supernatant is used for test.

B. Gewebe

It is recommended to take about 0.1 G Gewebe und hinzufügen 1 ml Extraktlösung, Volles Schleifen auf Eis.

Zentrifuge bei 8000 U/min für 10 Minuten bei 4 ℃, the supernatant is used for test.

C. Serum (Plasma) Probe: Detect sample

II. Bestimmung Verfahren

1. Spektrophotometer vorheizen 30 Protokoll, Wellenlänge anpassen 470 nm, Null mit destilliertem Setzen
2. Place Reagent I, Reagent II and Reagent III at 37℃ (Säugetier) oder 25 ℃(andere Arten) für 10 minutes before
3. Fügen Sie Reagenzien mit der folgenden Liste hinzu:

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. Berechnungen

ICH. Serum (Plasma)Probe

Einheitendefinition: 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).

POD(U/ml)=ΔA×Vrv÷Vsv÷0.01÷T =7133×ΔA

II. Gewebe, bacteria or culture cells

A. Proteinkonzentration

Einheitendefinition: 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.

POD(U/mg-Schutz)＝ΔA×Vrv÷(Vsv×Cpr)÷0.01÷T =7133×ΔA÷Cpr

B. Probengewicht

Einheitendefinition: 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.

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

C. CellAmount

Einheitendefinition: 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.

POD(U/104 -Zelle)＝ΔA×Vrv÷(500×Vsv÷Vs)÷0.01÷T =14.27×ΔA

Seil: Gesamtreaktionsvolumen, 1.07 ml; VSV: Gesamtüberstandsvolumen, 0.015 ml; Vs: Lösungsvolumen extrahieren, 1 ml;

T: Reaktionszeit, 1 Minute;

Cpr: Proteinkonzentration der Probe, mg/ml; W: Probengewicht, G;

500: Gesamtzahl von Bakterien oder Zellen, 5 Million.

Notiz:

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) oder 25 ℃ (andere Arten) for more than 10 Protokoll. 15 μL Probe und 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 Protokoll. Wenn ΔA größer ist als 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.
   

   Verweise

   • 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]. Analytische Biochemie, 1997,250(1):10-17.

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