Solarbio Succinate Déshydrogénase (SDH) Kit de test d'activité

Succinate déshydrogénase (SDH) Kit de test d'activité

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

Detection equipment: Spectrophotomètre

Chat non: BC0950

Taille: 50T/48S

Composants

Réactif I: 60 mL×1, grand à -20℃. Réactif II: 0.6 mL×1, grand à -20℃. Réactif III: 5 mL×1, store at 4℃.

Réactif IV: Poudre × 1, store at 4℃. When the solution will be used, add it into Reagent III to dissolve for use.

Réactif V: Poudre × 1, store at 4℃. Ajouter 4 mL d'eau distillée lorsque la solution sera utilisée, the unused reagents are stored at 4℃.

Réactif VI: Poudre × 1, grand à -20℃. Ajouter 3.333 mL d'eau distillée lorsque la solution sera utilisée, the unused reagents are stored at 4℃.

Description

Succinate déshydrogénase (SDH, CE 1.3.5.1) est largement trouvé chez les animaux, plantes, micro-organismes et cellules cultivées. SDH is a marker enzyme of mitochondria, which is a membrane binding enzyme located in the inner membrane of mitochondria. It is also one of the key points of respiratory electron transfer and oxidative phosphorylation. In addition, it provides electrons for the respiratory chain of various prokaryotic cells.

SDH can catalyze the dehydrogenation of succinic acid to fumaric acid. The dehydrogenation can reduce 2,6-dichlorophenol indophenol (DCPIP) under the transfer of phenazine dimethyl sulfate (PMS). 2,6- DCPIP has a characteristic absorption peak at 600 nm. The reduction rate of 2,6-DCPIP is determined by the change of absorbance at 600 nm, which represents the activity of SDH enzyme.

Required but not provided

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

Protocole

je. Extraction of SDH:

Accurately weigh 0.1 g of tissue or collect 5 millions de cellules, ajouter 1 mL of Reagent I and 10 µL de réactif II, homogenize by using homogenizer/mortar in ice bath, fully grind, centrifuger à 11000 ×g pour 10 minutes à 4℃, take the supernatant and place it on ice for testing.

II. Procédure

1. Preheat spectrophotometer for 30 minutes, ajuster la longueur d'onde à 600 nm et mettre à zéro avec de l'eau distillée.
2. Procedure test

Add each reagent to 1 mL glass cuvette in turn, and start timing at the same time of adding Reagent VI, record the initial absorbance A1 at the wavelength of 600 nm for 20 secondes. Then put the cuvette together with the reaction solution into a water bath of 37℃ (mammifère) ou 25 ℃ (autres espèces), and accurate reaction for 1 minute. Quickly take out the cuvette and dry it, and record the absorbance A2 at 80 seconds at 600 nm. ΔA = A1-A2, obtain ΔAT, ΔAB.

III. Calculation of SDH activity

Calculation formula for determination with 1 Cuvette en verre ml.

• Concentration en protéines

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every milligram tissue protein.

SDH(U/mg prot)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(Cpr×VS) ÷T＝1555.556×(ΔAT-ΔAB)÷Cpr

• Poids de l'échantillon

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every gram tissue.

SDH(U/g)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(VS÷VST×W) ÷T＝1571.111×(ΔAT-ΔAB)÷W

• Germ or cells

Définition de l'unité: One unit of enzyme activity is defined as the amount of enzyme catalyzes the consumed of 1 nmol of 2,6-dichlorophenol indophenol per minute in the reaction system every 10 thousand germ or cells.

SDH(Cellule U/104)＝[(ΔAT-ΔAB)÷(ε×d)×VRV×109]÷(VS÷VST×500) ÷T

＝3.142×(ΔAT-ΔAB)

VRT: Volume total de réaction, 0.98×10-3 L;

ε: The molar extinction coefficient of 2,6-DCPIP, 2.1×104 L/mol/cm; d: The light diameter of cuvette, 1 cm;

Contre: Volume d'échantillon, 0.03 ml;

VST: Add the volume of Reagent I and Reagent II, 1.01 ml; T: Temps de réaction(min), 1 minute;

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

500: Cells or germ, 5 million.

Note

1. All reagents and samples shall be placed on ice during the determination to avoid denaturation and deactivation.
2. If ΔA is greater than 0.5, the enzyme solution should be diluted with enzyme extract to obtain ΔA with less than 0.5, which can improve the detection sensitivity.
3. Because the Extract solution contains a certain concentration of protein (à propos 1 mg/ml), it is necessary to subtract the protein content of the Extract solution itself when determining the protein concentration of the sample.

Exemple expérimental:

1. Take 0.1g of kidney, ajouter 1 mL of Reagent Ⅰ and 10 μL Reagent Ⅱ, grind the homogenate with ice bath, centrifuge at 4℃ and 11000g for 10 min, and place the supernatant on ice. According to the determination procedure, the enzyme activity is calculated as follows: ΔAT= A1T- A2T=0.82-0.681=0.139, ΔAB= A1B- A2B=905-0.904=0.001

SDH activity (U/g masse) = 961.905×(ΔAT- ΔAB) ÷ W =2168.13 U/g mass.

Les références

• Fattoretti P, Bertoni-Freddari C, Caselli U, et autres. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging[J.]. Mechanisms of aging and development, 1998, 101(1-2): 175- 182.

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