成分:

萃取液: 80ml×1. 4℃保存.

試劑 1: 40ml×1. 4℃保存.

試劑 2: 粉末×1. -20℃保存, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. 稀 100 times when used.

試劑 3: 粉末×1. 4℃保存, dissolve with 3ml of acetone before use. 試劑 4: 5ml×1. 4℃保存.

產品描述:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, 植物, 微生物和培養細胞. 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 奈米, the activity of enzyme can be calculated by detecting the decrease rate of 2, 6-dichlorindolepheno.

需要但未提供的試劑和設備:

分光光度計, 水浴, 桌上型離心機, 移液管, 1ml glass cuvette, 砂漿/均化器, acetone, 冰和蒸餾水.

1. Complex II 萃取:

• Collecting 0.1g of tissue or 5 百萬個細胞, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 分分鐘. Discard the precipitate and transfer supernatant to another tube, 離心機在, 11000g and 4℃ for 15 分分鐘;
• The supernatant, IE。, cytoplasmic extract, can be used to determine the complex II leaking from mitochondria, this step can show the effect of mitochondrial extraction;
• 添加 400 μL extraction solution to sediment, splitting with ultrasonication (力量 20%, work time 5s, 間隔10秒, 重複 15 次), used to detect Complex Ⅱ activity and protein content.

Determining step

1. 預熱分光光度計 30 分分鐘, 調整波長至 605 奈米, 使用蒸餾將計數器設為零
2. 樣品測定

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 使用前. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (哺乳動物細胞) 或25℃(其他物種) 為了 15
• Add the following reagents in 1ml glass cuvette:

計算:

單位定義: 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/毫克蛋白質)=[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: light path of cuvette, 1 公分;

繩索: 總反應體積,1毫升; VS: 樣品量 (毫升), 0.05 毫升;

心肺復甦: 樣品蛋白濃度 (毫克/毫升); 時間: 反應時間 (分分鐘), 2 分分鐘;

筆記:

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, 乘以公式中的稀釋倍. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use 索拉生物 (PC0020 BCA蛋白 檢測試劑盒). 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. It is recommended to use the sample protein concentration to calculate the enzyme activity. If the sample fresh weight is used to calculate, the enzyme activity of cytoplasmic extract needs to be measured, and the sum of supernatant and precipitation enzyme activity is the total enzyme activity.
4. It’s enough for 50 tube reactions.
5. 依戀: 樣品重量(50T/24S)

• 上清液:

單位定義: 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;

繩索: 總反應體積,1毫升;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; d: light path of cuvette, 1 公分;

維: extract solution volume,1毫升; VS: 樣品量 (毫升), 0.05 毫升; 時間: 反應時間 (分分鐘), 2 分分鐘;

瓦: 樣品重量, G.

• Sediment:

單位定義: 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;

繩索: 總反應體積,1毫升;

e: 2, 6-dichlorindolepheno molar extinction coefficient, 2.1×104L/mol/cm; d: light path of cuvette, 1 公分;

維: sediment resuspended volume,0.4 毫升; VS: 樣品量 (毫升), 0.05 毫升;

時間: 反應時間 (分分鐘), 2 分分鐘; 瓦: 樣品重量, G.

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

實驗範例:

1. Take 0.1g of rabbit liver sample, 添加 1 萃取液毫升數, 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 (U/克質量) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 U/克質量

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

U/克質量

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

參考：

[1] Mühling J, Tiefenbach M, López-Barneo J, 等人. 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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