Componenti:

Estrarre la soluzione: 80ml×1. Conservazione a 4 ℃.

Reagente 1: 40ml×1. Conservazione a 4 ℃.

Reagente 2: Polvere×1. Conservazione a -20 ℃, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. Diluire 100 times when used.

Reagente 3: Polvere×1. Conservazione a 4 ℃, dissolve with 3ml of acetone before use. Reagente 4: 5ml×1. Conservazione a 4 ℃.

Descrizione del prodotto:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, pianta, microrganismi e cellule in coltura. It catalyzes succinic acid to form fumaric acid, reduce FAD to form FADH2. The FADH2 reduce oxidized CoQ to form reduced CoQ, which is a branch of respiratory electron transport chain.

CoQ that a catalytic product of complex II could reduce 2,6-dichloroindophenol, which has absorbance at 605 nm, the activity of enzyme can be calculated by detecting the decrease rate of 2, 6-dichlorindolepheno.

Reagenti e attrezzature necessari ma non forniti:

Spettrofotometro, bagnomaria, centrifuga da tavolo, pipetta di trasferimento, 1ml glass cuvette, mortaio/omogeneizzatore, acetone, ghiaccio e acqua distillata.

1. Complex II estrazione:

• Collecting 0.1g of tissue or 5 milioni di cellule, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 min. Discard the precipitate and transfer supernatant to another tube, centrifugare a, 11000ge 4℃ per 15 min;
• The supernatant, i.e., cytoplasmic extract, can be used to determine the complex II leaking from mitochondria, this step can show the effect of mitochondrial extraction;
• Aggiungere 400 μL extraction solution to sediment, splitting with ultrasonication (energia 20%, tempo di lavoro 5s, intervallo 10s, ripetere 15 volte), used to detect Complex Ⅱ activity and protein content.

Determining step

1. Preriscaldare lo spettrofotometro per 30 min, regolare la lunghezza d'onda su 605 nm, Imposta il contatore su zero con distillazione
2. Sample determination

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 prima dell'uso. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (cellula di mammifero) o 25 ℃(altre specie) per 15
• Add the following reagents in 1ml glass cuvette:

Calcolo:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno per mg of tissue protein in every minute.

Complex Ⅱ Activity (U/mg prot)=[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =476.2×ΔA÷Cpr

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: percorso luminoso della cuvetta, 1 cm;

Corda: volume totale di reazione,1ml; Contro: volume del campione (ml), 0.05 ml;

Cpr: concentrazione delle proteine ​​del campione (mg/ml); T: tempo di reazione (min), 2 min;

Nota:

1. Take two or three different samples for prediction before test to ensure the accuracy of experimental results. Dilute supernatant with distilled water if absorbance is higher than 1.5. Dilute sample with distilled water if ΔA>0.4, multiply dilute times in the formula. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use Solarbio (PC0020 Proteina BCA Kit di analisi). Because protein is contained in the extract, the protein content of the extract itself should be subtracted when determining the protein concentration of the sample.
3. Si consiglia di utilizzare la concentrazione proteica del campione per calcolare l'attività enzimatica. Se per il calcolo viene utilizzato il peso fresco del campione, è necessario misurare l'attività enzimatica dell'estratto citoplasmatico, e la somma dell'attività enzimatica del surnatante e della precipitazione costituisce l'attività enzimatica totale.
4. It’s enough for 50 reazioni del tubo.
5. Allegato: Peso del campione(50T/24S)

• Supernatante:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)=[ΔA1×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =476.2×ΔA1÷W ΔA1: supernatant absorbance;

Corda: volume totale di reazione,1ml;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: percorso luminoso della cuvetta, 1 cm;

Ve: extract solution volume,1ml; Contro: volume del campione (ml), 0.05 ml; T: tempo di reazione (min), 2 min;

W: peso del campione, G.

• Sedimento:

Definizione di unità: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1nmol of 2, 6-dichlorindolepheno in 1 min every gram of tissue weight.

Complex Ⅱ Activity(U/g)= [ΔA2×Vrv÷(ε×d)×109]÷(W÷Ve×Vs)÷T =190.5×ΔA2÷W ΔA2: sediment absorbance;

Corda: volume totale di reazione,1ml;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; D: percorso luminoso della cuvetta, 1 cm;

Ve: sediment resuspended volume,0.4 ml; Contro: volume del campione (ml), 0.05 ml;

T: tempo di reazione (min), 2 min; W: peso del campione, G.

• Total activity is the sum of ComplexⅡactivity in supernatant and sediment. Complex Ⅱ(U/g) =476.2×ΔA1÷W+190.5×ΔA2÷W.

Esempio sperimentale:

1. Take 0.1g of rabbit liver sample, aggiungere 1 ml di soluzione di estratto, grind and centrifuge the homogenate, and operate according to the determination steps. ΔA1 = A1-A2 = 1.134-1.054 = 0.08 in the supernatant, and ΔA2 =A1-A2 = 1.371-1.347 = 0.024 in the precipitation.

The activity of complex II in the supernatant (Massa U/g) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 Massa U/g

The activity of complex II in the precipitation (Massa U/g) = 190.5×ΔA2÷ W = 190.5×0.024÷0.1 = 45.72

Massa U/g

Complex II (Massa U/g) = 476.2× ΔA1÷W + 190.5×ΔA2÷W = 476.2×0.08÷0.1 + 190.5×0.024 ÷ 0.1 =426.8U/g mass.

Riferimenti：

[1] Mühling J, Tiefenbach M, López-Barneo J, et al. Mitochondrial complex II participates in normoxic and hypoxic regulation of α-keto acids in the murine heart[J]. Journal of molecular and cellular cardiology, 2010, 49(6): 950-961.

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