Соларбио Креатинкиназа (СК) Набор для анализа активности

Креатин киназа (СК) Набор для анализа активности

Примечание: Перед тестированием возьмите два или три разных образца для прогнозирования..

Операционное оборудование: Спектрофотометр

Кот нет: BC1140

Размер:50Т/48С

Компоненты:

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

Реагент I: powder×1, stored in dark at -20℃. Dissolved in 10 мл дистиллированной воды перед использованием, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Реагент II: powder×1, stored at -20℃. Dissolved in 0.5 мл дистиллированной воды перед использованием, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Реагент III: powder×2, stored at -20℃. Dissolved in 0.5 мл дистиллированной воды перед использованием, the reagents that cannot be used up shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Реагент IV: powder×1, stored at -20℃. Dissolved in 0.65 мл дистиллированной воды перед использованием, the unused reagent shall be stored at -20℃ after repacking, repeated freezing and thawing are prohibited.

Реагент V: 15 mL ×1. Хранение при 4℃.

Описание продукта:

Creatine kinase (СК) (ЕС 2.7.3.2) is also known as creatine phosphokinase, which mainly exists in heart, мышца, and brain. It can reversibly catalyze the trans-phosphorus reaction between creatine and ATP. It is an important kinase directly related to cell energy transport, muscle contraction and ATP regeneration.

CK catalyzes creatine phosphate and ADP to generate creatine and ATP, hexokinase catalyzes ATP and glucose to generate glucose-6-phosphate, and glucose-6-phosphate dehydrogenase catalyzes glucose-6- phosphate and NADP+ to generate NADPH, resulting in an increase of 340 nm light absorption value, which is used to express CK enzyme activity.

Требуемые реагенты и оборудование, которые не входят в комплект поставки

Scales, low temperature centrifuge, constant temperature water bath, спектрофотометр, 1 mL quartz cuvette and distilled water.

Процедура

я. Extraction of crude enzyme solution:

1. 1. Tissue sample:

The proportion of tissue mass (г): volume of Extract solution (мл): 1:5~10 (it is recommended to weigh about 0.1 г ткани, добавлять 1 mL of Extract solution) for ice bath homogeneity. Центрифуга в 10000 ×g для 15 минут при 4℃, возьмите супернатант и поместите его на лед для тестирования.

2. Образец сыворотки:

Direct determination.

3. Cell sample:

The number of cells (104): the volume of the Extract solution(мл) is 500~1000:1 (1 mL of Extract solution is recommended to be added to 5 миллион клеток), the Extract solution is added, and the cells are broken by ultrasonic wave in ice bath (Власть: 300Вт, ultrasonic: 3с, интервал: 7с, общее время: 3 минуты). Центрифуга в, 10000×g для 10 минут при 4℃, the supernatant and place it on ice for testing.

II. Test процедура:

1. Preheat the spectrophotometer for more than 30 минуты, отрегулировать длину волны, чтобы 340 нм, and adjust to zero with distilled water.
2. Рабочее решение: mix Reagent I, Реагент II, Реагент III, Reagent IV and Reagent V in the proportion of 70:4:7:10:90 (volume ratio) перед использованием. Prepare when the solution will be used. Incubate for 20 minutes in the room temperature before use (this step cannot be omitted).
3. Операционный стол: add the following reagents into 1 mL cuvette

III. Calculation of СК:

• Calculated by tissue protein concentration:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmolof NADPH per minute at 37℃ and pH7.0 every milligram of protein.

CK activity (Ед/мг прот) = ΔA÷(ε×d)×VRT×109 ÷ (VS× Cpr) ÷ T= 268 ×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 catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 in every gram of sample.

CK activity (U/g fresh weight) = ΔA÷(ε×d)×VRT×109 ÷ (ВС÷ВСТ×В) ÷T= 268×ΔA÷W

• Calculated by serum volume:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 in every milliliter of serum.

CK activity (Ед/мл) = ΔA÷(ε×d)×VRV×109 ÷ VS÷T= 268×ΔA

• By cell count:

Definition of enzyme activity: One unit of enzyme activity is defined as the amount of enzyme catalyze the production of 1 nmol of NADPH per minute at 37℃ and pH7.0 every 10000 клетки.

CK activity (U/104cell)=ΔA÷(ε×d)×VRV×109÷(VS ÷ VST×N)÷T=268 ×ΔA÷ N

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

ВРТ: Total volume of reaction system, 0.001 мл;

VS: The volume of sample in reaction system, 0.2 мл; VST: The volume of extract solution, 1 мл;

КПР: Концентрация белка образца, мг/мл; Вт: The mass of sample mass, г;

Н: The number of cells, 104 единицы; Т: reaction time, 3 минуты.

Примечание:

1. The CK of serum is not stable. The samples are collected and measured as soon as possible. The CK of serum is stable for 24 hours after being stored at 4℃ in dark.
2. The protein content of the sample needs to be determined separately. белок БСА content determination kit can be used for determination.
3. If the OD value is greater than 0.6, the sample can be diluted properly with the extract solution, and calculation formula can be changed according dilution ratio.
4. ΔAB generally does not exceed 01.

Экспериментальные экземпляры：

1. Take 0.1g of mouse brain, добавьте 1 мл раствора экстракта, homogenate and grind. Возьмите супернатант, then dilute with extract 4 times and detect according to the measured steps. Calculate ΔAT=A2T- A1T=0.638-0.149=0.489, ΔAB=A2B-A1B=0， ΔA=ΔAT-ΔAB=0.489-0=0.489, рассчитать активность фермента в зависимости от веса образца:

CK activity（ U/g weight） =268×ΔA÷W×4（ dilution ratio） =268×0.489÷0.1×4（ dilution ratio）

=5242.08U/g weight.

2. Take 200μL serum of duck to detect directly, calculate ΔAT=A2T-A1T=0.445-0.423=0.022, ΔAB=A2B- A1B=0, ΔA=ΔAT-ΔAB=0.022-0=0.022, calculate the enzyme activity according to volume of serum:

CK activity（U/mL）=268×ΔA=268×0.022=5.896 U/mL.

Рекомендации：

• Defang Li, Ning Lu, JichunHan, et al.Eriodictyol Attenuates Myocardial Ischemia-Reperfusion Injury through the Activation of JAK2. Frontiers in Immunology. January2018;(IF3.845)
• Xu Y, Meng X, Hou X, и другие. A mutant of the ButhusmartensiiKarsch antitumor-analgesic peptide exhibitsreducedinhibition to hNav1. 4 and hNav1. 5 channels while retaining analgesic activity[Дж].Journal of Biological Chemistry, 2017, 292(44):18270-18280

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