Solarbio Phosphoenolpyruvate Carboxykinase (PEPCK) Набор для анализа активности

Атрибут	Подробности
Примечание	Перед тестированием возьмите два или три разных образца для прогнозирования.
Операционное оборудование	Спектрофотометр
Кот нет	BC3310
Размер	50Т/48С

Компоненты:

Экстракт раствора: 60 мл ×1. Хранение при 4℃.

Реагент I: 45 мл×1, stored at 4℃.

Реагент II: Порошок × 1, stored at -20℃ and protected from light. Dissolve it in 35 mL of Reagent I before use. The reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

Реагент III: 45 мкл × 1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 1:120, to prepare when the solution will be used.

Реагент IV: 155 мкл × 1, stored at 4℃ and protected from light. Before temporary use, add distilled water to dilute according to the volume ratio of 7:250, to prepare when the solution will be used.

Реагент V: Порошок×1, stored at -20℃ and protected from light. Dissolve it in 2.5 мл дистиллированной воды перед использованием, the reagent that cannot be used up shall be stored at -20℃ after repacking, repeat freezing and thawing are prohibited;

Описание продукта:

PEPCK (ЕС 4.1.1.32) широко встречается у животных, flowering plants, algae, some fungi, and bacteria. The enzyme catalyzes the conversion of oxaloacetic acid to phosphoenolpyruvate, which is the first-rate limiting enzyme regulating gluconeogenesis.

PEPCK catalyzes oxaloacetic acid to form phosphoenolpyruvate and CO2, pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD+ in turn, and determine the NADH decline rate at 340 нм, which can reflect the PEPCK activity.

Требуемые реагенты и оборудование, которые не входят в комплект поставки

Спектрофотометр, Низкая температура центрифуга, water bath pot, 1 мл кварцевая кювета, регулируемая пипетка, ступка/гомогенизатор, лед и дистиллированная вода

Процедура

я. Extraction of crude enzyme solution:

1. 1. Образец ткани:

The proportion of tissue mass (г): volume of extract solution (мл): 1:5~10 (рекомендуется взвесить около 0.1 г ткани, добавлять 1 mL of extract solution) for ice bath homogenate, then centrifugate at 8000 ×g для 10 минут при 4℃, возьмите супернатант и поместите его на лед для тестирования.

2. Бактерии или культивируемые клетки:

Первый, collect bacteria or cells into the centrifuge tube, and then discard the supernatant. The number of cells (104): the volume of the extract (мл) является 500-1000:1 (1 mL of the extract solution is recommended to be added to 5 миллионов бактерий или клеток), and the cells are broken by ultrasonic wave in ice bath (Власть: 200W or 20%, ultrasonic:3с, интервал:10с, Повторить 30 раз). Then centrifuged at 8000 ×g для 10 минут при 4℃, возьмите супернатант и поместите его на лед для тестирования.

3. Образец сыворотки: Direct determination.

II. Тест процедура:

1. Preheat the spectrophotometer for more than 30 минуты, отрегулировать длину волны, чтобы 340 нм, and adjust to zero with distilled
2. Рабочее решение: mix Reagent II, Реагент III, Reagent IV in the proportion of 7:1:1(В:В:В) перед использованием. Prepare when the solution will be used.
3. Preheat the working solution at 37℃ (млекопитающее) или 25 ℃ (другие виды) для 5 минуты.
4. Операционный стол: Add the following reagents to the 1 mL quartz cuvette in turn:

Название реагента (мкл)	Пустая трубка(Б)	Пробирка(Т)
Образец	—	50
Дистиллированная вода	50
Рабочее решение	900	900
Реагент V	50	50
Add Reagent V and mix it immediately. Measure the absorbance value A1 at 340 nm for 10s and A2 at 70s. Calculate ΔAT= A1T- A2T, ΔAB= А1В- А2Б, and ΔA =ΔAT-ΔAB. Blank tube only needs to be done once or twice.

III. Calculation of PEPCK:

1. Calculation by micro quartz cuvette

• Calculated by tissue protein concentration:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, каждый миллиграмм белка.

PEPCK activity (Ед/мг прот) = ΔA÷(ε×d)×VRT×109 ÷ (VS× Cpr) ÷ T =3215.4×ΔA÷Cpr

• Calculated by the quality of tissue samples:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute, каждый грамм образца.

PEPCK activity (U/g свежий вес) = ΔA÷(ε×d)×VRT×109 ÷ (ВС÷ВСТ×В) ÷T= 3215.4×ΔA÷W

• By cell count:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every 10 thousand cells

PEPCK activity (Ячейка U / 104)=ΔA÷(ε×d)×VRV×109÷(ВС÷ВСТ×500)÷T=6.43×ΔA

• Calculated by serum volume:

Definition of enzyme activity: one unit of enzyme activity is defined as the amount of enzyme that catalyzes the consumption of 1 nmol of NADH per minute every milliliter of serum

PEPCK activity (Ед/мл) = ΔA÷(ε×d)×VRV×109÷VS÷T=3215.4×ΔA

е: Molar extinction coefficient of NADH, 6.22×103 L/mol/cm; д: Light diameter of cuvette, 1 см;

ВРТ: Total volume of reaction system, 0.001 л;

ПРОТИВ: The volume of sample in reaction system, 0.05 мл; VST: The volume of extract solution, 1 мл;

КПР: Концентрация белка образца, мг/мл, Self-determination of protein concentration; Вт: The mass of sample mass, г;

Т: Время реакции, 1 минуты;

500: Общее количество бактерий или клеток, 5 миллион; 109: Unit conversion factor, 1 моль = 109 НМОЛ.

Примечание:

1. When A1 is less than 1 or ΔA is greater than 0.6, it is recommended to dilute the sample to a proper multiple before determination to improve the detection
2. For samples with high enzyme activity, such as animal liver, kidney and other tissues, it is recommended to dilute the extract to 5 times or more for
3. The blank tube is a test well for testing the quality of each reagent component. Under normal conditions, the change does not exceed06.
4. The steps of sample adding and mixing shall be rapid, and the stopwatch timing shall be accurate. If conditions permit, it is recommended that two persons cooperate to complete this

Экспериментальный пример:

1. Take 0.1g liver and add 1 ml extract solution for homogenate. Take the supernatant and dilute it twice with the extract solution. Then operate according to the determination steps. Measure and calculate: ΔAT= A1D- A2D= 1.175-0.532=0.643, ΔAB= А1В — A2B =1.29-1.244=0.046, ΔA = ΔAT — ΔAB = 0.643-0.046 "=" 0.597

PEPCK activity (Ед/г массы) = 3215.4×ΔA÷W×10 (коэффициент разбавления) = 3215.4×0.597÷0.1×10=191959.4 U/g mass.

2. Take 0.1g aloe vera and add 1 ml extract solution for homogenization, take the supernatant and operate according to the determination steps. Measure and calculate ΔAT = A1T- A2T =1.257-1.106=0.151, ΔAB= А1В- A2B =1.29-1.244=0.046, ΔA = ΔAT — ΔAB =0.151-0.046=0.105

PEPCK activity (Ед/г массы) "=" 3215.4 × ΔA÷W =3215.4×0.105÷0.1=3376.17 U/g mass.

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