구성요소:

용액 추출: 80ml×1. 4℃에서 보관.

시약 1: 40ml×1. 4℃에서 보관.

시약 2: 가루×1. -20℃에서 보관, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. 묽게 한 100 times when used.

시약 3: 가루×1. 4℃에서 보관, dissolve with 3ml of acetone before use. 시약 4: 5ml×1. 4℃에서 보관.

제품 설명:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, plant, 미생물과 배양세포. 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.

필요하지만 제공되지 않는 시약 및 장비:

분광 광도계, 욕조, 책상 원심분리기, 이송 피펫, 1ml glass cuvette, 모르타르/균질화기, acetone, 얼음과 증류수.

1. Complex II extraction:

• Collecting 0.1g of tissue or 5 백만개의 세포, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 분. Discard the precipitate and transfer supernatant to another tube, 원심분리기, 11000g and 4℃ for 15 분;
• The supernatant, 즉., cytoplasmic extract, can be used to determine the complex II leaking from mitochondria, this step can show the effect of mitochondrial extraction;
• 추가하다 400 μL extraction solution to sediment, splitting with ultrasonication (힘 20%, work time 5s, 간격 10초, 반복하다 15 타임스), used to detect Complex Ⅱ activity and protein content.

Determining step

1. 예열 분광 광도계 30 분, 파장을 조정하여 605 nm, 증류수로 카운터를 0으로 설정
2. 샘플 결정

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 사용하기 전에. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (mammal cell) 또는 25℃(다른 종) ~을 위한 15
• Add the following reagents in 1ml glass cuvette:

계산:

단위 정의: 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 프로트)=[ΔA×Vrv÷(ε×d)×109]¶(VS×심폐소생술)÷T =476.2×ΔA÷Cpr

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

로프: total reaction volume,1밀리리터; 대: 샘플량 (밀리리터), 0.05 밀리리터;

심폐소생술: 샘플 단백질 농도 (mg/mL); 티: 반응 시간 (분), 2 분;

메모:

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 솔라바이오 (PC0020 BCA 단백질 분석 키트). 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. 부착: 샘플 중량(50T/24S)

• 상청액:

단위 정의: 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]¶(W²Ve×Vs)÷T =476.2×ΔA1÷W ΔA1: supernatant absorbance;

로프: total reaction volume,1밀리리터;

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

베: extract solution volume,1밀리리터; 대: 샘플량 (밀리리터), 0.05 밀리리터; 티: 반응 시간 (분), 2 분;

여: 샘플 중량, g.

• Sediment:

단위 정의: 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]¶(W²Ve×Vs)÷T =190.5×ΔA2÷W ΔA2: sediment absorbance;

로프: total reaction volume,1밀리리터;

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

베: sediment resuspended volume,0.4 밀리리터; 대: 샘플량 (밀리리터), 0.05 밀리리터;

티: 반응 시간 (분), 2 분; 여: 샘플 중량, g.

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

실험예:

1. Take 0.1g of rabbit liver sample, 추가하다 1 mL의 추출 용액, 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 질량) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 U/g 질량

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

U/g 질량

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

참고자료：

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

관련 상품：

BC0510/BC0515 Electron Transport Chain Complex I Activity Assay Kit

BC3240/BC3245 Electron transport chain Complex Ⅲ Activity Assay Kit

BC0940/BC0945 Electron transport chain Complex Ⅳ Activity Assay Kit

BC1440/BC1445 Electron transport chain Complex Ⅴ Activity Assay Kit