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抽出液: 80ml×1. 4℃で保存.

試薬 1: 40ml×1. 4℃で保存.

試薬 2: パウダー×1. -20℃で保管, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. 希薄 100 times when used.

試薬 3: パウダー×1. 4℃で保存, dissolve with 3ml of acetone before use. 試薬 4: 5ml×1. 4℃で保存.

製品説明:

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

必要だが提供されていない試薬と装置:

分光光度計, 水浴, 卓上遠心分離機, 移送ピペット, 1ml glass cuvette, mortar/ homogenizer, acetone, 氷と蒸留水.

1. Complex II extraction:

• Collecting 0.1g of tissue or 5 百万個の細胞, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 分. Discard the precipitate and transfer supernatant to another tube, で遠心分離する, 11000g and 4℃ for 15 分;
• 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;
• 追加 400 μL extraction solution to sediment, splitting with ultrasonication (力 20%, 作業時間5秒, 間隔10秒, 繰り返す 15 回), used to detect Complex Ⅱ activity and protein content.

Determining step

1. 予熱用分光光度計 30 分, 波長を調整して 605 nm, set the counter to zero with distilled
2. Sample determination

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 使用前に. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (哺乳類細胞) または25℃(他の種) のために 15
• Add the following reagents in 1ml glass cuvette:

計算:

単位の定義: 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/mgプロット)=[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 cm;

ロープ: 総反応量,1mL; 対: サンプル量 (mL), 0.05 mL;

心肺蘇生法: サンプルタンパク質濃度 (mg/mL); T: reaction time (分), 2 分;

注記:

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, multiply dilute times in the formula. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use ソーラーバイオ (PC0020 BCAプロテイン アッセイキット). 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. 酵素活性を計算するにはサンプルタンパク質濃度を使用することをお勧めします。. サンプルの新鮮重量を計算に使用する場合, 細胞質抽出物の酵素活性を測定する必要がある, 上清と沈殿酵素活性の合計が総酵素活性となります。.
4. It’s enough for 50 チューブ反応.
5. アタッチメント: サンプル重量(50T/24S)

• 上清:

単位の定義: 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;

ロープ: 総反応量,1mL;

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

ヴェ: extract solution volume,1mL; 対: サンプル量 (mL), 0.05 mL; T: reaction time (分), 2 分;

W: サンプル重量, g.

• 堆積物:

単位の定義: 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;

ロープ: 総反応量,1mL;

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

ヴェ: sediment resuspended volume,0.4 mL; 対: サンプル量 (mL), 0.05 mL;

T: reaction time (分), 2 分; W: サンプル重量, g.

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

実験例:

1. Take 0.1g of rabbit liver sample, 追加 1 抽出液 mL, 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/g質量) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 U/g質量

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

U/g質量

Complex II (U/g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.

参考文献：

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