บันทึก: ใช้ตัวอย่างที่แตกต่างกันสองหรือสามตัวอย่างเพื่อทำนายก่อนการทดสอบ. อุปกรณ์การดำเนินงาน: High-performance liquid chromatography หมายเลขแค็ตตาล็อก: BC5114
Sizes：50ที/48ส
ส่วนประกอบ:
Extract solution I: 80 มล.×1. Storage at 2-8℃.
Extract solution II：40 มล.×1. Storage at 2-8℃.
รีเอเจนต์ I: 15 มล.×1. Storage at 2-8℃. ก่อนใช้งาน, เอา 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.
รีเอเจนต์ II:10 ml × 1. Storage at 2-8℃.
ATP Standard： ผง×1. การจัดเก็บที่ -20 ℃. ก่อนใช้งาน, 1.8 mL distilled water is added to prepare 1 ไมโครโมล / mL ATP standard solution, which was frozen at -20 องศาเซลเซียส. To ensure the integrity of ATP, avoid repeated freezing and thawing.
ADP Standard： ผง×1. การจัดเก็บที่ -20 ℃. ก่อนใช้งาน, 2.34 mL distilled water is added to prepare 1 ไมโครโมล / mL ATP standard solution, which was frozen at -20 องศาเซลเซียส. To ensure the integrity of ATP, avoid repeated freezing and thawing.
AMP Standard： ผง×1. การจัดเก็บที่ -20 ℃. ก่อนใช้งาน, 2.0 mL distilled water is added to prepare 1 ไมโครโมล / mL ATP standard solution, which was frozen at -20 องศาเซลเซียส. To ensure the integrity of ATP, avoid repeated freezing and thawing.
รายละเอียดสินค้า：
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, พืช, microorganisms, และเซลล์เพาะเลี้ยง. 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) พบมากในสัตว์, พืช, microorganisms, และเซลล์เพาะเลี้ยง. 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 นาโนเมตร, and their content can be determined by high-performance liquid chromatography.
รีเอเจนต์และอุปกรณ์ที่จำเป็นแต่ไม่ได้จัดเตรียมไว้ให้：
High-efficiency liquid chromatograph (C18 column (4.6×250 mm), ultraviolet detector (VWD)), เครื่องหมุนเหวี่ยงเดสก์ท็อป, ปิเปตแบบปรับได้, mortar/ homogenizer, brown EP tube, 50 syringe filters (น้ำ, 0.45
µm), syringe, suction filter, filter membrane (organic, น้ำ), 50 brown injection bottle (2 มล), อะซีโตไนไตรล์ (chromatographically pure, 500 มล), ultrapure water.
Preparations before the experiment:
- ใช้ 500 mL of chromatographically pure acetonitrile (mobile phase A) และ 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).
- 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
- Preparation of ATP standard: 1 μmol/mL ATP standard solution is diluted with distilled water to 0.5 ไมโครโมล/มล, 0.1 ไมโครโมล/มล, 0.05 ไมโครโมล/มล, 0.01 ไมโครโมล/มล, 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).
- Preparation of ADP standard: 1 μmol/mL ADP standard solution is diluted with distilled water to 0.5 ไมโครโมล/มล, 0.1 ไมโครโมล/มล, 0.05 ไมโครโมล/มล, 0.01 ไมโครโมล/มล, 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).
- Preparation of AMP standard: 1 μmol/mL AMP standard solution is diluted with distilled water to
0.5 ไมโครโมล/มล, 0.1 ไมโครโมล/มล, 0.05 ไมโครโมล/มล, 0.01 ไมโครโมล/มล, 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).
ขั้นตอน
1. การจัดเตรียมตัวอย่าง:
-
- ตัวอย่างเนื้อเยื่อ: According to the ratio of tissue (ก): extract solution I (มล) - 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 นาที. เครื่องปั่นแยกที่ 10000 รอบต่อนาทีสำหรับ 10 min at 4°C, take 750μL of the supernatant, add 750μL of extract II, shake well (5 นาที) and mix well, เครื่องหมุนเหวี่ยงอีกครั้งที่ 10000 rpm at 4°C for 10 นาที, and take the supernatant to filter with an aqueous syringe filter into the brown injection bottle to be tested at room temperature (ภายใน 2 ชม.).
- ตัวอย่างเซลล์: According to the ratio of 10 ล้านเซลล์ (หน่วย): extract solution I (มล)= 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 วินาที, interval of 7 วินาที, total time of 3 นาที); centrifuge at 4℃, 10000 รอบต่อนาทีสำหรับ 10 นาที, take 750μL of the supernatant, add 750μL of extract II, shake well (5 นาที) and mix well, เครื่องหมุนเหวี่ยงอีกครั้งที่ 10000 rpm at 4°C for 10 นาที, and take the supernatant to filter with an aqueous syringe filter into the brown injection bottle to be tested at room temperature (within 2h).
- เซรั่ม: It is recommended to take 0.4 mL serum sample, เพิ่ม 0.6 mL extraction solution 1, and extract for 40 min in an ice bath. เครื่องปั่นแยกที่ 10000 รอบต่อนาทีสำหรับ 10 min at 4°C, take 750μL of the supernatant, add 750μL of extract II, shake well (5 นาที) and mix well, เครื่องหมุนเหวี่ยงอีกครั้งที่ 10000 rpm at 4°C for 10 นาที, 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. การกำหนด ขั้นตอน：
- 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: 27องศาเซลเซียส, flow rate 0.8 มล./นาที, and wavelength 254 นาโนเมตร, the elution program is as shown in the table below, and the sampling time is 70 นาที. After setting, save the method group.
- 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.
- Detect the prepared standard solution, the injection volume is 10 ไมโครลิตร, the ATP, ADP, and AMP can be separated within 10 นาที, and the retention time of ATP, ADP, and AMP is about 7.8 นาที, 6.7min and 5.4min (the pH of the system, คอลัมน์, mobile phase, ฯลฯ. are different, the retention time is different, only reference).
- Detect the prepared sample solution, the injection volume is 10 ไมโครลิตร, and detect the peak area of ATP, ADP, and AMP at the corresponding retention
| เวลา | Mobile Phase | ||
| ก | บี | ||
| 0 นาที | 2% | 98% | |
| 10 นาที | 2% | 98% | |
| 15 นาที | 70% | 30% | |
| 50 นาที | 70% | 30% | |
| 55 นาที | 2% | 98% | |
| 70 นาที | 2% | 98% |
3. การคำนวณ：
- Draw standard curves of ATP, ADP, AMP with the standard concentration (µmol/mL) 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 (ไมโครโมล/มล) in the
ATP content การคำนวณ
- น้ำหนักตัวอย่าง
ATP (ไมโครโมล/กรัม)＝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, 1.5มล; ว: น้ำหนักตัวอย่าง, ก; MATP: 551.14; 2: Sample dilution factor.
- Liquidvolume:
ATP (ไมโครโมล/มล)＝2 x1 ×VE÷VS=5×x1
ATP (μg/mL）=2 x1 ×VE×551.14 ÷VS =2755.7 ×x1
VE: volume of extract solution I, 1มล; MATP: 551.14; VS： volume of sample, 0.4มล； 2: Sample dilution factor.
- จำนวนเซลล์
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, 1มล; MATP: 551.14; เอ็น: number of cells, 104 as a unit; 2: Sample dilution factor.
ADP content การคำนวณ
- น้ำหนักตัวอย่าง
ADP (ไมโครโมล/กรัม)＝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, 1.5มล; ว: น้ำหนักตัวอย่าง, ก; MADP: 427.2; 2: Sample dilution factor.
- Liquidvolume:
ADP (ไมโครโมล/มล)＝2 x2 ×VE÷VS=5×x2
ADP (μg/mL）=2 x2 ×VE×427.2÷VS =2136×x2
VE: volume of extract solution I, 1มล; MADP: 427.2; VS： volume of sample, 0.4มล； 2: Sample dilution factor.
- จำนวนเซลล์
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, 1มล; MADP: 427.2; เอ็น: number of cells, 104 as a unit; 2: Sample dilution factor.
AMP content การคำนวณ
- น้ำหนักตัวอย่าง
AMP (ไมโครโมล/กรัม)＝2 x3×VE÷W=3 x3÷W
AMP (μg/g）=2 x3 ×VE×499.19÷W=1497.57×3۞W
VE: volume of extract solution I, 1.5มล; ว: น้ำหนักตัวอย่าง, ก; MAMP: 499.19; 2: Sample dilution factor.
- Liquidvolume:
AMP (ไมโครโมล/มล)＝2 x3 ×VE÷VS=5×x3
AMP (μg/mL）=2 x3 ×VE×499.19÷VS =2495.95×x3
VE: volume of extract solution I, 1มล; MAMP: 499.19; VS： volume of sample, 0.4มล； 2: Sample dilution factor.
- จำนวนเซลล์
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, 1มล; MAMP: 499.19; เอ็น: number of cells, 104 as a unit; 2: Sample dilution factor.
บันทึก:
ข้อควรระวัง:
- After the detection, the chromatographic column needs to be flushed with high-concentration ultrapure water (เกี่ยวกับ 20-30 column volumes) to prevent clogging the chromatographic column. ในที่สุด, flush the column according to the specifications of the column to prevent damage to the chromatographic
- 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
- The sample after extraction is not stable at room temperature, so it needs to be operated as soon as possible.
- 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
รีวิว
ยังไม่มีบทวิจารณ์