Kit de test d'activité Solarbio Ca++Mg++-ATPase

Californie++Mg++-ATPase Activity Assay Kit

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

Équipement d'exploitation: Spectrophotomètre

Chat non: BC0960

Taille: 50T/24S

Composants:

Réactif I: Liquide 30 mL×1. Stockage à 4℃.

Réactif II: Liquide 4 mL×1. Stockage à 4℃.

Réactif III: Poudre × 2. Stockage à -20℃. Dissolve thoroughly with 1 mL d'eau distillée avant utilisation. The rest reagent can be kept at -20℃ for one week.

Réactif IV: Liquide 2 mL×1. Stockage à 4℃.

Réactif V: Poudre × 1. Stockage à 4℃. Dissolve thoroughly with 3 mL d'eau distillée avant utilisation. Réactif VI: Poudre × 1. Stockage à 4℃. Dissolve thoroughly with 15 mL d'eau distillée avant utilisation, can be kept at 4℃ for one week.

Reagent VII: Poudre × 1. Stockage à 4℃. Dissolve thoroughly with 15 mL d'eau distillée avant utilisation, can be kept at 4℃ for one week.

Reagent VIII: Liquide 15 mL×1. Storage at RT.

Solution standard: Liquide 1 mL×1. 10 μmol/mL standard phosphorus liquid, stockage à 4℃.

0.5 μmol/mL standard phosphorus working solution: Dilute the 10 μmol/mL standard 20 times with distilled water to 0.5 μmol/mL standard. Par exemple: ajouter 1.9 mL d'eau distillée pour 0.1 mL of standard, bien mélanger.

Phosphorus fixing reagent:

Prepare reagents for determining phosphorus content: make solution as the volume ratio of H2O: Réactif VI: Reagent VII: Reagent VIII =2:1:1:1, which should be light yellow. It shows lose efficacy if color is changed, phosphorus pollution if color is change to blue. Prepare the reagent when it will be used.

Note: It is better to use new beakers, glass rods and glass pipettes or disposable plastic ware when making reagent to avoid phosphorus pollution.

Description du produit:

Ca++Mg++-ATPase is widely distributed in plants, animaux, microorganisms and cells, which catalyzes the hydrolysis of ATP to form ADP and inorganic phosphorus.

Ca++Mg++-ATPase decomposes ATP to produce ADP and inorganic phosphorus. The activity of ATPase can be detected by measuring the amount of inorganic phosphorus.

Réactifs et équipements requis mais non fournis:

Spectrophotomètre, centrifugeuse de bureau, pipette réglable, bain d'eau, 1 Cuvette en verre ml, mortier/homogénéisateur, glace et eau distillée.

Procédure:

je. La préparation des échantillons:

1. Bactéries ou cellules:

Collecting bacteria or cells into a centrifuge tube, centrifugation, and discard supernatant. Suggest add 1mL of Reagent I to 5 million of bacteria or cells. Use ultrasonic to splitting bacteria and cells (placé sur la glace, ultrasonic power 20%, working time 3 secondes, interval 10 secondes, répéter pour 30 fois). Centrifuger à 8000 ×g pour 10 minutes at 4℃ and take the supernatant on ice before testing.

2. Tissu:

Ajouter 1 mL of Reagent I into 0.1 g of tissue, broyer complètement sur la glace. Centrifuger à 8000 ×g pour 10 minutes at 4℃ and take the supernatant on ice before testing.

3. Sérum: Directly

II. Détermination:

1. Preheat spectrophotometer for 30 minutes, ajuster la longueur d'onde à 660 nm, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:

3. Determination of phosphorus content, add the following reagents to 1.5 mL EP tube:

Bien mélanger, then place the mix solution in a 40℃water bath for 10 minutes. Cooling to room temperature and detect the absorbance at 660 nm. The blank tube and standard tube just need one or two tubes.

III. Calcul:

1. Sérum:

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milliliter of serum.

Ca++Mg++-ATPase (U/mL)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷s÷T

=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]

2. Tissu, bactéries, ou cellules

• Concentration en protéines:

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milligram of tissue protein.

Ca++Mg++-ATPase (U/mg prot)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs × Cpr)÷T

=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]÷Cpr

• Poids de l'échantillon:

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour, every milligram of tissue.

Ca++Mg++-ATPase (U/g poids)=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs÷V1×W)÷T

=7.5×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]÷W

• bacteria or cells

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every 10000 cells or bacteria.

Ca++Mg++-ATPase (U/104cellule )=Cs×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]×Vrv÷(Vs÷V1×500)÷T

=0.015×[ΔA(T)-ΔA(C)]÷[ΔA(S)-ΔA(B)]

Cs: Concentrate of standard tube, 0.5 µmol/mL;

Corde: Volume total de réaction, 0.5 ml; Contre: Volume d'échantillon, 0.2 ml;

Cpr: Concentration de protéines de l'échantillon (mg/ml); T: Temps de réaction (min), 1/6 heure;

W: Poids de l'échantillon (g);

Vl: Volume of reagent I, 1 ml;

500: The amount of bacteria or cell, 5 million.

Note

1. This kit can detect 24 tubes of Ca++Mg++ -ATPase samples in 50 tubes for each sample need one tube as control.
2. This method has the characteristics of trace, sensitive and rapid. The test tubes used for determination are phosphate-free strictly. Avoiding phosphorus pollution is the key to the success of detection.

Exemple expérimental:

1. Take of pancreas and add 1 mL of Reagent I for ice bath homogenization. After centrifugation at 4℃ for 10 min, the supernatant is put on the ice and operated according to the determination steps. ΔAT = 0.916-0.389=0.527, ΔAS =0.398-0.004=0.394

Ca++Mg++- ATPase activity (U/g masse) = 7.5 × ΔAT ÷ΔAS ÷ W = 100.32 U/g masse.

2. Take 0.1g of willow and add 1 mL of Reagent Ⅰ for ice bath homogenization. After centrifugation at 4℃ for 10 min, the supernatant is put on ice and operated according to the determination steps. The ΔAT=0.137-0.124=0.013, and the ΔAS=398-0.004=0.394

Californie + + Mg + + – ATPase activity (U/g masse) = 7.5×ΔAT ÷ ΔAS ÷ W = 2.47 U/g masse.

Les références

[1] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J.]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2008, 1777(4): 362-368.

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