Компоненты:

Экстракт раствора: 80ml×1. Хранение при 4℃.

Реагент 1: 40ml×1. Хранение при 4℃.

Реагент 2: Порошок×1. Хранение при -20℃, dissolve with 1ml of acetone. The dissolved reagent 2 can be stored at -20℃ after dispensing. Dilute 100 times when used.

Реагент 3: Порошок×1. Хранение при 4℃, dissolve with 3ml of acetone before use. Реагент 4: 5ml×1. Хранение при 4℃.

Описание продукта:

Mitochondrial complex II is the same as succinate-Co-enzyme Q reductase, which exists widely in mitochondria of animal, растение, микроорганизмы и культивируемые клетки. 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 нм, the activity of enzyme can be calculated by detecting the decrease rate of 2, 6-dichlorindolepheno.

Требуемые реагенты и оборудование, которые не входят в комплект поставки:

Спектрофотометр, водяная баня, настольная центрифуга, пипетка для переноса, 1ml glass cuvette, ступка/гомогенизатор, acetone, лед и дистиллированная вода.

1. Complex II добыча:

• Collecting 0.1g of tissue or 5 миллион клеток, add 1ml extract solution and grind on ice with mortar/homogenizer;
• centrifuge at 600g and 4℃for 10 мин. Discard the precipitate and transfer supernatant to another tube, центрифуга в, 11000g and 4℃ for 15 мин;
• 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;
• Добавлять 400 μL extraction solution to sediment, splitting with ultrasonication (власть 20%, work time 5s, интервал 10 с, repeat 15 раз), used to detect Complex Ⅱ activity and protein content.

Determining step

1. Предварительно разогрейте спектрофотометр для 30 мин, отрегулировать длину волны, чтобы 605 нм, обнулить счетчик дистиллированной
2. Sample determination

• Making working solution: mix reagent 2 and reagent 3 according to 1:1 перед использованием. Prepare when used. Prepared when the solution will be used.
• Preheat reagent1 at 37℃ (mammal cell) или 25℃(другие виды) для 15
• Add the following reagents in 1ml glass cuvette:

Расчет:

Определение единицы измерения: 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 (Ед/мг прот)"="[ΔA×Vrv÷(ε×d)×109]÷(Vs×Cpr)÷T =476.2×ΔA÷Cpr

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

Веревка: total reaction volume,1мл; Против: объем пробы (мл), 0.05 мл;

КПР: концентрация белка в образце (мг/мл); Т: reaction time (мин), 2 мин;

Примечание:

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, умножьте разбавление в формуле. Increase sample volume if ΔA is slow.
2. Detect sample protein concentrate by yourself, you can use Соларбио (PC0020 БСА-белок Набор для анализа). 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. Вложение: Вес образца(50Т/24С)

• Супернатант:

Определение единицы измерения: 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(Ед/г)"="[ΔA1×Vrv÷(ε×d)×109]÷(Вт÷Ve×Vs)÷T =476.2×ΔA1÷W ΔA1: supernatant absorbance;

Веревка: total reaction volume,1мл;

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

ве: extract solution volume,1мл; Против: объем пробы (мл), 0.05 мл; Т: reaction time (мин), 2 мин;

Вт: sample weight, г.

• Sediment:

Определение единицы измерения: 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(Ед/г)"=" [ΔA2×Vrv÷(ε×d)×109]÷(Вт÷Ve×Vs)÷T =190.5×ΔA2÷W ΔA2: sediment absorbance;

Веревка: total reaction volume,1мл;

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

ве: sediment resuspended volume,0.4 мл; Против: объем пробы (мл), 0.05 мл;

Т: reaction time (мин), 2 мин; Вт: sample weight, г.

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

Экспериментальный пример:

1. Take 0.1g of rabbit liver sample, добавлять 1 mL of Extract solution, 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 (Ед/г массы) = 476.2×ΔA1÷W = 476.2 × 0.08÷0.1 = 380.96 Ед/г массы

The activity of complex II in the precipitation (Ед/г массы) = 190.5×ΔA2÷ W = 190.5×0.024÷0.1 = 45.72

Ед/г массы

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

Рекомендации：

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

сопутствующие товары：

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