Solarbio ATP ADP AMP Content HPLC Assay Kit 50T | 48С

Примечание: Перед тестом возьмите два или три разных образца для прогнозирования.. Операционное оборудование: High-performance liquid chromatography Номер каталога: BC5114

Sizes：50Т/48С

Компоненты:

Extract solution I: 80 мл×1. Storage at 2-8℃.

Extract solution II：40 мл×1. Storage at 2-8℃.

Реагент I: 15 мл×1. Storage at 2-8℃. Перед использованием, брать 3.5 mL of reagent I and add it to 1000 mL of ultrapure water, adjust its pH to 6.15 with reagent II to form mobile phase B, and seal it.

Реагент II:10 mL ×1. Storage at 2-8℃.

ATP Standard： Порошок×1. Хранение при -20℃. Перед использованием, 1.8 mL distilled water is added to prepare 1 мкмоль / mL ATP standard solution, which was frozen at -20 °С. To ensure the integrity of ATP, avoid repeated freezing and thawing.

ADP Standard： Порошок×1. Хранение при -20℃. Перед использованием, 2.34 mL distilled water is added to prepare 1 мкмоль / mL ATP standard solution, which was frozen at -20 °С. To ensure the integrity of ATP, avoid repeated freezing and thawing.

AMP Standard： Порошок×1. Хранение при -20℃. Перед использованием, 2.0 mL distilled water is added to prepare 1 мкмоль / mL ATP standard solution, which was frozen at -20 °С. To ensure the integrity of ATP, avoid repeated freezing and thawing.

Описание продукта：

Nucleotides have important biological functions. They are a class of compounds composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphoric acid. They are mainly involved in the formation of nucleosides.

Adenosine triphosphate (ATP) is considered to be a universal energy source that is essential for cell synthesis in the survival and reproduction of all organisms. ATP can be produced through a variety of cellular pathways. The most typical example is synthesis by adenosine triphosphate synthase through oxidative phosphorylation in mitochondria, or synthesis by photosynthesis in plant chloroplasts. The main energy sources for ATP synthesis are glucose and fatty acids.

Adenosine diphosphate (ADP) is widely present in animals, растения, микроорганизмы, и культивируемые клетки. In organisms, ADP is a product of breaking a high-energy phosphate bond (ATP) hydrolyzed to lose a phosphate radical) and releasing energy.

Adenosine monophosphate (AMP) широко встречается у животных, растения, микроорганизмы, и культивируемые клетки. It is formed after ATP and ADP release energy in the body. It can continue to bind phosphate groups to form adenosine diphosphate (ADP) and adenosine triphosphate (ATP). It is the product of incomplete hydrolysis of ATP.

ATP 、 ADP 、 AMP have an absorption peak at 254 нм, and their content can be determined by high-performance liquid chromatography.

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

High-efficiency liquid chromatograph (C18 column (4.6×250 mm), ultraviolet detector (VWD)), настольная центрифуга, регулируемая пипетка, mortar/ homogenizer, brown EP tube, 50 syringe filters (вода, 0.45

µm), syringe, suction filter, filter membrane (organic, вода), 50 brown injection bottle (2 мл), acetonitrile (chromatographically pure, 500 мл), ultrapure water.

Preparations before the experiment:

1. Использовать 500 mL of chromatographically pure acetonitrile (mobile phase A) и 1000 mL of configured mobile phase B to filter with a filter membrane to remove impurities in the solvent to prevent clogging the chromatographic column. (Acetonitrile uses a 0.45 µm organic filter membrane for suction filtration, and the configured mobile phase B uses a 0.22 µm aqueous filter membrane for suction filtration).
2. Ultrasound the prepared mobile phases A and B for 30 minutes to remove the gas in the solvent to prevent clogging the chromatographic column and affecting the experimental
3. Preparation of ATP standard: 1 μmol/mL ATP standard solution is diluted with distilled water to 0.5 мкмоль/мл, 0.1 мкмоль/мл, 0.05 мкмоль/мл, 0.01 мкмоль/мл, 0.005 μmol/mL ATP standard solution. (The prepared standard concentration is for reference only and can be adjusted according to the actual sample concentration). The standard is filtered using an aqueous syringe filter into the brown injection bottle to be tested (please place it at room temperature before testing, so as not to affect the retention time).
4. Preparation of ADP standard: 1 μmol/mL ADP standard solution is diluted with distilled water to 0.5 мкмоль/мл, 0.1 мкмоль/мл, 0.05 мкмоль/мл, 0.01 мкмоль/мл, 0.005 μmol/mL ADP standard solution. (The prepared standard concentration is for reference only and can be adjusted according to the actual sample concentration). The standard is filtered using an aqueous syringe filter into the brown injection bottle to be tested (please place it at room temperature before testing, so as not to affect the retention time).
5. Preparation of AMP standard: 1 μmol/mL AMP standard solution is diluted with distilled water to

0.5 мкмоль/мл, 0.1 мкмоль/мл, 0.05 мкмоль/мл, 0.01 мкмоль/мл, 0.005 μmol/mL AMP standard solution. (The prepared standard concentration is for reference only and can be adjusted according to the actual sample concentration). The standard is filtered using an aqueous syringe filter into the brown injection bottle to be tested (please place it at room temperature before testing, so as not to affect the retention time).

Процедура

1. Базовые приготовления:

• • Tissue sample: According to the ratio of tissue (г): extract solution I (мл) "=" 1:5~10 (it is recommended to weigh 3 g tissue sample and add 1.5 mL extract solution I) to add extract solution I, homogenate on ice, and extract in an ice bath for 40 мин. Центрифуга в 10000 об/мин для 10 min at 4°C, take 750μL of the supernatant, add 750μL of extract II, хорошо встряхнуть (5 мин) and mix well, centrifuge again at 10000 rpm at 4°C for 10 мин, and take the supernatant to filter with an aqueous syringe filter into the brown injection bottle to be tested at room temperature (within 2 час).
  • Cell sample: According to the ratio of 10 миллион клеток (единицы): extract solution I (мл)= 1000~500:1 (it is recommended to take 10 million cell samples and add 1 mL extract solution I) add extraction solution I, ultrasonic breaking Cells on ice (power 300W, ultrasound for 3 секунды, interval of 7 секунды, total time of 3 минуты); centrifuge at 4℃, 10000 об/мин для 10 минуты, take 750μL of the supernatant, add 750μL of extract II, хорошо встряхнуть (5 мин) and mix well, centrifuge again at 10000 rpm at 4°C for 10 мин, and take the supernatant to filter with an aqueous syringe filter into the brown injection bottle to be tested at room temperature (within 2h).
  • сыворотка: It is recommended to take 0.4 mL serum sample, добавлять 0.6 mL extraction solution 1, and extract for 40 min in an ice bath. Центрифуга в 10000 об/мин для 10 min at 4°C, take 750μL of the supernatant, add 750μL of extract II, хорошо встряхнуть (5 мин) and mix well, centrifuge again at 10000 rpm at 4°C for 10 мин, and take the supernatant to filter with an aqueous syringe filter into the brown injection bottle to be tested at room temperature (within 2h).

2. Определение процедура：

1. Turn on the computer, turn on the switch buttons of each module of the HPLC, install the chromatographic column, open the software, and set the injection volume in the method group to 10µL, column temperature: 27°С, flow rate 0.8 mL/min, and wavelength 254 нм, the elution program is as shown in the table below, and the sampling time is 70 мин. After setting, save the method group.
2. Clean the column with the mobile phase, equilibrate the column with a mobile phase ratio of acetonitrile: mobile phase B (pH = 6.15) "=" 2: 98, and start the injection after the baseline is stable.
3. Detect the prepared standard solution, the injection volume is 10 мкл, the ATP, ADP, and AMP can be separated within 10 минуты, and the retention time of ATP, ADP, and AMP is about 7.8 мин, 6.7min and 5.4min (the pH of the system, column, mobile phase, и т. д.. are different, the retention time is different, only reference).
4. Detect the prepared sample solution, the injection volume is 10 мкл, and detect the peak area of ATP, ADP, and AMP at the corresponding retention

3. Расчеты：

1. Draw standard curves of ATP, ADP, AMP with the standard concentration (мкмоль/мл) as x and the peak area as the y. Substitute the peak area of the sample into the standard curve to calculate the ATP, ADP, AMP concentration x1, x2, x3 (мкмоль/мл) in the

ATP content calculation

1. Вес образца

ATP (μmol/g)＝2 x1×VE÷W=3 x1÷W

ATP (μg/g）=2 x1 ×VE×551.14 ÷W=1653.42 x1÷W

VE: volume of extract solution I, 1.5мл; Вт: Вес образца, г; MATP: 551.14; 2: Sample dilution factor.

1. Liquidvolume:

ATP (мкмоль/мл)＝2 x1 ×VE÷VS=5×x1

ATP (μg/mL）=2 x1 ×VE×551.14 ÷VS =2755.7 ×x1

VE: volume of extract solution I, 1мл; MATP: 551.14; VS： volume of sample, 0.4мл； 2: Sample dilution factor.

1. Количество ячеек

ATP (μmol/104 cell)＝2 x1 ×VE÷ N =2×x1÷ N

ATP (μg/g104 cell）=2 x1 ×VE×551.14 ÷N =1102.28×x1÷N

VE: volume of extract solution I, 1мл; MATP: 551.14; Н: number of cells, 104 as a unit; 2: Sample dilution factor.

ADP content calculation

1. Вес образца

ADP (μmol/g)＝2 x2×VE÷W=3 x2÷W

ADP (μg/g）=2 x2 ×VE×427.2 ÷W=1281.6 x2÷W

VE: volume of extract solution I, 1.5мл; Вт: Вес образца, г; MADP: 427.2; 2: Sample dilution factor.

1. Liquidvolume:

ADP (мкмоль/мл)＝2 x2 ×VE÷VS=5×x2

ADP (μg/mL）=2 x2 ×VE×427.2÷VS =2136×x2

VE: volume of extract solution I, 1мл; MADP: 427.2; VS： volume of sample, 0.4мл； 2: Sample dilution factor.

1. Количество ячеек

ADP (μmol/104 cell)＝2 x2 ×VE÷N =0.002×x2÷N

ADP (μg/g104 cell）=2 x2 ×VE×427.2 ÷N =854.4×x2÷N

VE: volume of extract solution I, 1мл; MADP: 427.2; Н: number of cells, 104 as a unit; 2: Sample dilution factor.

AMP content calculation

1. Вес образца

AMP (μmol/g)＝2 x3×VE÷W=3 x3÷W

AMP (μg/g）=2 x3 ×VE×499.19÷W=1497.57×3÷Вт

VE: volume of extract solution I, 1.5мл; Вт: Вес образца, г; MAMP: 499.19; 2: Sample dilution factor.

1. Liquidvolume:

AMP (мкмоль/мл)＝2 x3 ×VE÷VS=5×x3

AMP (μg/mL）=2 x3 ×VE×499.19÷VS =2495.95×x3

VE: volume of extract solution I, 1мл; MAMP: 499.19; VS： volume of sample, 0.4мл； 2: Sample dilution factor.

1. Количество ячеек

AMP (μmol/104 cell)＝2 x3 ×VE÷N =2×x3÷N

AMP (μg/g104 cell）=2 x3 ×VE×499.19 ÷N =998.38×x3÷N

VE: volume of extract solution I, 1мл; MAMP: 499.19; Н: number of cells, 104 as a unit; 2: Sample dilution factor.

Примечание:

Меры предосторожности:

1. After the detection, the chromatographic column needs to be flushed with high-concentration ultrapure water (о 20-30 column volumes) to prevent clogging the chromatographic column. Окончательно, flush the column according to the specifications of the column to prevent damage to the chromatographic
2. The dilution factor of the standard should be determined according to the concentration of nucleotides in the sample. The peak area of nucleotides in the sample must be within the peak area of the standard solution of different concentrations. The dilution factor of the standard is only a reference. If the nucleotide concentration in the sample is too high, it is recommended to dilute it before
3. The sample after extraction is not stable at room temperature, so it needs to be operated as soon as possible.
4. If the sample number is too large, it is recommended to test the standard solution once a day (one standard solution is sufficient) to determine the corresponding retention