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Coenzyme I NAD (H) content test kit visible spectrophotometry method
NegotiableUpdate on 02/19
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Overview
Related products of visible spectrophotometry for coenzyme I NAD (H) content test kit: 6-phosphogluconate dehydrogenase (G6PDH)/glucose 6-phosphogluconate dehydrogenase test kit 6-phosphogluconate dehydrogenase (G6PDH)/glucose 6-phosphogluconate dehydrogenase test kit microcalorimetry 6-phosphogluconate dehydrogenase (G6PDH)/glucose 6-phosphogluconate dehydrogenase test kit 6-phosphogluconate dehydrogenase (G6PDH)/glucose 6-phosphogluconate dehydrogenase test kit UV spectrophotometry Cytoplasmic isocitrate dehydrogenase (ICDHc) test kit Cytoplasmic isocitrate dehydrogenase (ICDHc) test kit microcalorimetry
Product Details
Special reminder: This product is only for scientific research use and should not be used for direct clinical testing on the human body.
Product Name:Coenzyme I NAD (H) content test kit visible spectrophotometry method
Product specifications: 50 tubes/24 samples
Detection method: Visible spectrophotometry
Product Code: BK-01S6322
Product Category:
Product Introduction:
| Measurement significance Coenzyme I NAD (H) is widely present in animals, plants, microorganisms, and cultured cells. NAD+is the main hydrogen receptor for glycolysis (EMP) and tricarboxylic acid cycle (TCA), and the generated NADH is transferred to oxygen through the respiratory electron chain (ETC) to form a large amount of ROS while synthesizing ATP. At the same time, NADH is regenerated into NAD+. The oxidation reactions involved in the breakdown of the three major metabolites of sugar, lipids, and proteins are mostly carried out through this system. The levels of NAD (H) content and NADH/NAD+ratio can be used to evaluate the strength of glycolysis and TCA cycle. A higher NAD (H) and NADH/NAD+ratio indicates a higher oxygen consumption in cellular respiration and a state of peroxidation. In addition, an increase in the NADH/NAD+ratio can also inhibit glycolysis and TCA cycle. In addition, NAD+degradation products play important regulatory roles in cellular signaling, metabolism, and gene expression. Measurement principle Extract NAD+and NADH from the sample using acidic and alkaline extraction solutions, respectively. NADH is reduced to MTT by PMS hydrogenation, and the absorbance value is detected at 570nm; And NAD+can be reduced to NADH by ethanol dehydrogenase, further detected by MTT reduction method. Equipment and supplies that need to be self provided Visible spectrophotometer, desktop centrifuge, pipette, 1 mL glass colorimetric dish, mortar, ice, and distilled water. |
Required instruments and supplies:
Visible spectrophotometer, 1mL glass cuvette (optical path 1cm), low-temperature centrifuge, pipette, mortar, ice, and distilled water
4、 Determination of Superoxide Dismutase (SOD):
Suggest selecting 2 samples for prediction before the formal experiment, understanding the situation of this batch of samples, familiarizing oneself with the experimental process, and avoiding experiments
Waste of samples and reagents!
1. Sample preparation
|
① Organizational sample: Take about 0.1g of tissue (0.25g for samples with sufficient moisture), add 1mL of extraction solution, and perform the extraction at 4 º C or in an ice bath Homogenize (or use various common homogenizers). Centrifuge at 4 º C × 12000rpm for 10 minutes, and take the supernatant as the test solution. [Note]: If the sample size is increased, extraction can be carried out in a ratio of tissue mass (g) to extraction solution volume (mL) of 1:5-10 |
② Bacterial/cellular samples: Collect bacteria or cells into a centrifuge tube first, centrifuge and discard the supernatant; Take about 5 million bacteria or cells and add them to 1mL Extract solution, sonicate bacteria or cells (ice bath, power 200W, sonication for 3s, interval 10s, repeated 30 times); Centrifuge at 12000rpm at 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing. [Note]: If the sample size is increased, it can be based on the number of bacteria/cells (10) 4) Extract in a ratio of 500-1000:1 using an extraction solution (mL). ③ Liquid sample: direct detection; If turbid, centrifuge and take the supernatant for detection. |
Experimental Methodology:
1、 Preparation of Heparin:
Commercial heparin freeze-dried powder 140 units/mg, packaged in 1 gram bottles, with 140000 units per bottle. Weigh 0.1 grams of heparin freeze-dried powder and add 5ml of physiological saline to prepare 2800 units/ml. Take 50-100 μ l of moistened tube wall, which can anticoagulate 3-5ml of human blood without coagulation. Mouse blood is prone to clotting, so it's best to make it thicker. You can take 0.1 grams of heparin freeze-dried powder, dissolve it in 3ml of physiological saline, and take 50-100 μ l, which can anticoagulate 2-4ml of mouse blood.
Preparation of heparin anticoagulant tube: Take 0.1 grams of heparin freeze-dried powder and add 2.5 ml of physiological saline. Take 100 μ l to 150 μ l and drop it into a plastic or glass tube, moisten the tube wall, and place it in a small oven below 80 ℃ (you can use a small oven for baking bread). Rotate it horizontally every 5 to 10 minutes until it is dry. Store it at 4 ℃ to prevent 2-5 ml of blood from clotting.
2、 Collection of blood samples:
Whole blood is divided into two types based on the collection conditions: non anticoagulant and anticoagulant. Meanwhile, the upper layer of yellow liquid separated without anticoagulation is called serum; The upper layer of yellow liquid separated by anticoagulation is called plasma.
1. Non anticoagulant serum collection: Collect whole blood, let it stand for 1-2 hours, and centrifuge at low speed to separate the serum for later use or storage.
2. Anticoagulation plasma collection: Collect anticoagulated whole blood, gently invert and fully anticoagulate, and then centrifuge the plasma directly at low speed (or let it stand for about half an hour before centrifugation at low speed) for later use or storage. Select the appropriate anticoagulant based on its performance characteristics. The commonly used anticoagulants in the laboratory include various salts of heparin, EDTA, and others.
Points to note when choosing anticoagulation:
① The amount of anticoagulant added to each sample should be consistent, and the amount of whole blood taken should also be as consistent as possible;
② After collecting anticoagulated whole blood, it is necessary to gently invert it and fully anticoagulate it to prevent some blood from not coming into contact with anticoagulants and causing coagulation;
③ A relatively large amount of plasma is collected from anticoagulated whole blood (0.4-0.5ml of plasma can be separated from 1ml of anticoagulated whole blood);
④ After the plasma collected for anticoagulation is frozen and stored, there may be flocculent turbidity during thawing. If there is turbidity, it needs to be removed by centrifugation
Used for measurement.
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Notes:
1. Reagent 2 is an enzyme and cannot be frozen. It should be placed on ice during use.
2. Only one care is needed.
3. If the absorbance value of the control tube is greater than 2, it is recommended to dilute reagent 2 with distilled water by 7 times before use (10 μ l of reagent 2 original solution+60 μ l of distilled water).
4. Why are some sample measuring tubes larger than the control tube for SOD, and what is the range of values for the control tube?
The scope of care is 0.8-2. The low absorbance value of the control tube may be due to (1) reagent two or reagent four not being prepared and used immediately; (2) Not adding reagents in order; (3) The reaction time is not sufficient, and can be extended (reaction time of 30 minutes can be extended to 40 minutes). The high absorbance value of the control tube may be due to reagent 2 not being diluted by the corresponding multiple according to the operating instructions.
If the measuring tube is larger than the control tube, it may be due to the significant influence of impurities in the sample. In order to reduce the influence of impurities, the sample extraction supernatant is usually diluted 10 times with distilled water or extraction solution before testing, which can usually make the measurement normal. Multiply by the corresponding dilution factor in the calculation formula.
