Peroxydase (COSSE) Kit de test d'activité

Note: Prenez deux ou trois échantillons différents pour la prédiction avant le test.

Équipement d'exploitation: Spectrophotomètre Numéro de catalogue: BC0090 Sizes：50T/48S

Composants:

Extraire la solution: 60 mL×1. Stockage à 4℃.

Réactif I: 40 mL×1. Stockage à 4℃.

Réactif II: 0.5 mL ×1. Stockage à 4℃. Centrifuge before use. Prendre 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.

Réactif III: 10 mL×1. Stockage à 4℃.

Description du produit

Peroxydase (COSSE, CE 1.11.1.7) widely exists in animals, plantes, et micro-organismes. 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.

Réactifs et équipements requis mais non fournis

Spectrophotomètre, centrifugeuse de bureau, transferpettor, 1 Cuvette en verre ml, mortier/homogénéisateur, glace et eau distillée.

Procédure

je. La préparation des échantillons:

UN. Bactéries ou cellules

Collecte de bactéries ou de cellules dans le tube à centrifuger, the supernatant is discarded after centrifugation. It is suggested to take about 5 millions de bactéries/cellule et ajoutez 1 mL de solution d'extrait. Bacteria or cell is splitted by ultrasonication (Pouvoir: 20% ou 200 W, temps de travail 3s, intervalle 10s, répéter pour 30 fois). Centrifuger à 8000 tr/min et 4℃ pour 10 minutes, the supernatant is used for test.

B. Tissu

It is recommended to take about 0.1 g de mouchoir et ajouter 1 mL de solution d'extrait, broyer complètement sur la glace.

Centrifuger à 8000 tr/min pour 10 minutes à 4℃, the supernatant is used for test.

C. Sérum (plasma) échantillon: Detect sample

II. Détermination procédure

1. Preheat spectrophotometer for 30 minutes, ajuster la longueur d'onde à 470 nm, mettre à zéro avec distillé
2. Place Reagent I, Reagent II and Reagent III at 37℃ (mammifère) ou 25 ℃(autres espèces) pour 10 minutes before
3. Ajoutez des réactifs avec la liste suivante:

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. Calculs

je. Sérum (plasma)échantillon

Définition de l'unité: 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).

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

II. Tissu, bacteria or culture cells

UN. Concentration en protéines

Définition de l'unité: 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.

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

B. Poids de l'échantillon

Définition de l'unité: 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.

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

C. Montant de la cellule

Définition de l'unité: 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.

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

Corde: Volume total de réaction, 1.07 ml; Vsv: Volume total de surnageant, 0.015 ml; Contre: Extraire le volume de la solution, 1 ml;

T: Temps de réaction, 1 minute;

Cpr: Concentration de protéines de l'échantillon, mg/ml; W: Poids de l'échantillon, g;

500: Nombre total de bactéries ou de cellules, 5 million.

Note:

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) ou 25 ℃ (autres espèces) for more than 10 minutes. 15 µL d'échantillon et 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 minutes. 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.
   

   Les références

   • 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.]. Biochimie analytique, 1997,250(1):10-17.

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