ATP酶广泛分布于植物、动物、微生物和细胞的生物膜酶系统中，具有载体和酶的功能，可以维 持细胞膜电位和调节渗透压，并为营养物质的吸收提供动力，对维持细胞正常生理功能起着极其重要的作用，并且可作为细胞能量代谢及功能有无损伤的重要评价指标。
检测原理：Na⁺/K⁺-ATP酶（Na⁺/K⁺-ATPase）能够分解ATP生成ADP和无机磷，无机磷在酸性条件下能够与钼酸铵反应生成磷钼酸铵，经还原后生成蓝色磷钼蓝，产物在660nm处具有特征吸收峰，通过吸光值的变化即可表征Na⁺/K⁺-ATP酶的活性。
适用样本：血清（浆）、动植物组织、细胞、细菌
N1505953
| Component
| 48T | 96T | Storage
|
N1505953A
| Extraction Buffer
| 60 mL
| 60 mL×2
| 2-8℃ |
N1505953B
| Assay Buffer
| 5 mL
| 10 mL
| 2-8℃
|
N1505953C
| Reagent Ⅰ
| 1EA | 1EA
| -20℃. Store in the dark. |
N1505953D
| Reagent Ⅱ
| 1 mL
| 2 mL | 2-8℃
|
N1505953E
| Reagent Ⅲ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953F
| Reagent Ⅳ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953G
| Reagent Ⅴ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953H
| Reagent Ⅵ
| 25 mL | 25 mL
| 2-8℃
|
N1505953I
| Standard
| 5 mL
| 10 mL
| 2-8℃ |
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
酶标仪或可见光分光光度计（能测660nm处的吸光度）
恒温箱
96孔板或微量玻璃比色皿、可调节式移液枪及枪头
去离子水
匀浆器（如果是组织样本）
实验流程
1、试剂准备
试剂名称
| 试剂准备
| 注意事项
|
Extraction Buffer
| 即用型；使用前平衡到室温
| 4℃保存
|
Assay Buffer
| 即用型；使用前平衡到室温
| 4℃保存
|
Working Reagent Ⅰ
| 临用前加7.2mL去离子水溶解
| 用不完的试剂-20℃避光分装保存1周，避免反复冻融
|
Reagent Ⅱ
| 即用型；使用前平衡到室温
| 4℃保存
|
Working Reagent Ⅲ
| 临用前加3mL去离子水溶解
| 用不完的试剂4℃避光保存可稳定2周
|
Working Reagent Ⅳ
| 临用前加25mL去离子水溶解
| 用不完的试剂4℃避光保存可稳定2周
|
Working Reagent Ⅴ
| 临用前加25mL去离子水溶解
| 用不完的试剂4℃避光保存可稳定2周
|
Reagent Ⅵ
| 即用型；使用前平衡到室温
| 4℃保存
|
Standard
| 即用型；使用前平衡到室温
| 4℃保存
|
0.5μmol/mL标准磷应用液
| 将Standard以20倍稀释，即取0.1mL Standard加1.9mL去离子水充分混匀。
|
|
定磷剂
| 按去离子水: Working Reagent Ⅳ: Working Reagent Ⅴ: Reagent Ⅵ=2:1:1:1的比例配制，配好的定磷剂应为浅黄色。
| 若无色则试剂失效，若是蓝色则为磷污染，定磷剂现配现用。 |
注意：
(1) Reagent Ⅲ有刺激性气味，Reagent Ⅴ有毒，Reagent Ⅵ有腐蚀性，建议在通风橱进行实验。
(2) 配试剂最好用新的烧杯、玻璃棒和玻璃移液器，也可以用一次性塑料器皿避免磷污染。
2、样本制备
注意：
(1) 推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存1个月。处理好的样本须当天检测。
(2) 如需测定蛋白浓度，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
2.1 血浆、血清等液体样本
可直接检测。
2.2 动物组织样本
按0.1g组织加入1mL的Extraction Buffer的比例进行冰浴匀浆。匀浆液于4℃，8,000g离心10min。取上清，置于冰上待测。
2.3 细菌和细胞样本
收集细菌或细胞到离心管内，4℃，12,000g离心1min弃上清；按照每5×10<sup>6</sup>个细菌或细胞加入1 mL Extraction Buffer，超声波破碎细菌或细胞（功率20%或200W，超声3s，间隔10s，重复30次）；4℃，8,000g离心10min，取上清，置冰上待测。
2.4 植物样本
用冷的PBS冲洗植物样本去除表面的杂质。按0.1 g组织加入1 mL 的Extraction Buffer的比例进行冰浴匀浆。匀浆液于4℃，8,000g离心10min。取上清，置于冰上待测。
3、实验步骤
3.1 酶标仪或可见分光光度计预热30min以上，调节波长至660nm，可见分光光度计用去离子水调零。
3.2 酶促反应（在EP管中加入下列试剂）
试剂 (μL)
| 对照管
| 测定管
|
Assay Buffer
| 65
| 45 |
Working Reagent Ⅰ
| 60 | 60 |
Reagent Ⅱ
| 0 | 20 |
样本
| 0 | 100 |
混匀，置于37℃（哺乳动物）或25℃（其他物种）水浴锅准确反应10min；
Working Reagent Ⅲ
| 25
| 25
|
样本
| 100
| 0 |
混匀，4,000g，常温离心10min，取上清液。
3.3 定磷（96孔板或微量玻璃比色皿中加入下列试剂）
试剂 (μL)
| 空白孔
| 标准孔
| 对照孔
| 测定孔
|
0.5μmol/mL 标准磷应用液
| 0 | 20
| 0
| 0
|
上清液
| 0
| 0
| 20
| 20
|
去离子水
| 20
| 0
| 0
| 0
|
定磷剂
| 200
| 200
| 200
| 200
|
3.4 混匀，室温放置30min，在660nm处，记录各管吸光值。
注意：每个样本均需要做对照孔，空白孔和标准孔各做1个即可。实验之前建议选择2-3个预期差异大的样本做预实验。如果A测定-A对照大于0.3，样本可用Extraction Buffer进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
4、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
4.1 血清（浆）等液体样本Na⁺/K⁺-ATPase活力的计算
定义：规定每小时每毫升血清（浆）中Na⁺/K⁺-ATPase分解ATP产生1 μmol无机磷的量为一个酶活力单位。
Na⁺/K⁺-ATPase活力 (U/mL)=C标准×(A测定-A对照)÷(A标准-A空白)×V总÷V样÷(T÷60)=7.5×(A测定-A对照)÷(A标准-A空白)
4.2 组织、细菌或细胞中Na⁺/K⁺-ATPase活力的计算
(1) 按蛋白浓度计算
定义：规定每小时每毫克组织蛋白中Na⁺/K⁺-ATPase分解ATP产生1 μmol无机磷的量为一个酶活力单位。
Na⁺/K⁺-ATPase活力 (U/mg)=C标准×(A测定-A对照)÷(A标准-A空白)×V总÷(Cpr×V样)÷(T÷60)=7.5×(A测定-A对照)÷(A标准-A空白)÷Cpr
(2) 按样本鲜重计算
定义：规定每小时每克组织中Na⁺/K⁺-ATPase分解ATP产生1 μmol无机磷的量为一个酶活力单位。
Na⁺/K⁺-ATPase活力 (U/g)=C标准×(A测定-A对照)÷(A标准-A空白)×V总÷(V样÷V样总×W)÷(T÷60)=7.5×(A测定-A对照)÷(A标准-A空白)÷W
(3) 按细菌或细胞密度计算
定义：规定每小时每1万个细菌或细胞中Na⁺/K⁺-ATPase分解ATP产生1 μmol无机磷的量为一个酶活力单位。
Na⁺/K⁺-ATPase活力 (U/10<sup>4</sup>)=C标准×(A测定-A对照)÷(A标准-A空白)×V总÷(V样÷V样总×500)÷(T÷60)=0.015×(A测定-A对照)÷(A标准-A空白)
参数说明：
C标准：标准管浓度，0.5 μmol/mL；
V总：酶促反应总体积，0.25 mL；
V样：加入样本体积，0.1 mL；
V样总：加入Extraction Buffer体积，1 mL；
T：反应时间，10 min；
Cpr：样本蛋白质浓度，mg/mL；
W：样本鲜重，g；
500：细菌或细胞总数，500万个。
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、如需测定蛋白浓度，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3、生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护， 穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
4、本产品仅供科学研究使用，不适用于临床诊断。
ATPase is widely distributed in the biological membrane enzyme systems of plants, animals, microorganisms, and cells. It functions as both a carrier and an enzyme, maintaining the cell membrane potential, regulating osmotic pressure, and providing the driving force for nutrient absorption. It plays an extremely important role in maintaining normal cellular physiological functions and can serve as a key evaluation indicator for cellular energy metabolism and functional integrity.
Detection Principle: Na⁺/K⁺-ATPase decomposes ATP to generate ADP and inorganic phosphorus. Under acidic conditions, inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate, which is then reduced to form a blue phosphomolybdenum complex. This product has a characteristic absorption peak at 660 nm. The activity of Na⁺/K⁺-ATPase can be characterized by the change in absorbance.
Applicable Samples: Serum (plasma), animal and plant tissues, cells, bacteria
N1505953
| Component
| 48T | 96T | Storage
|
N1505953A
| Extraction Buffer
| 60 mL
| 60 mL×2
| 2-8℃ |
N1505953B
| Assay Buffer
| 5 mL
| 10 mL
| 2-8℃
|
N1505953C
| Reagent Ⅰ
| 1EA | 1EA
| -20℃. Store in the dark. |
N1505953D
| Reagent Ⅱ
| 1 mL
| 2 mL | 2-8℃
|
N1505953E
| Reagent Ⅲ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953F
| Reagent Ⅳ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953G
| Reagent Ⅴ
| 1EA
| 1EA
| 2-8℃. Store in the dark.
|
N1505953H
| Reagent Ⅵ
| 25 mL | 25 mL
| 2-8℃
|
N1505953I
| Standard
| 5 mL
| 10 mL
| 2-8℃ |
Note: Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.
Required Instruments and Reagents
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 660 nm)
Incubator
96-well plate or micro glass cuvettes, adjustable pipettes and tips
Deionized water
Homogenizer (for tissue samples)
Experimental Procedure
1. Reagent Preparation
Reagent Name
| Reagent Preparation
| Notes
|
Extraction Buffer
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
Assay Buffer
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
Working Reagent Ⅰ
| Dissolve in 7.2 mL deionized water immediately before use.
| Unused reagent can be aliquoted and stored at -20℃ protected from light for 1 week; avoid repeated freeze-thaw cycles.
|
Reagent Ⅱ
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
Working Reagent Ⅲ
| Dissolve in 3 mL deionized water immediately before use.
| Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
|
Working Reagent Ⅳ
| Dissolve in 25 mL deionized water immediately before use.
| Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
|
Working Reagent Ⅴ
| Dissolve in 25 mL deionized water immediately before use.
| Unused reagent can be stored at 4℃ protected from light for up to 2 weeks.
|
Reagent Ⅵ
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
Standard
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
0.5 μmol/mL Standard Phosphorus Application Solution
| Dilute the Standard 20-fold: Take 0.1 mL Standard and add 1.9 mL deionized water, mix thoroughly.
|
|
Phosphorus Assay Reagent
| Prepare by mixing deionized water : Working Reagent Ⅳ : Working Reagent Ⅴ : Reagent Ⅵ = 2 : 1 : 1 : 1. The prepared reagent should be light yellow.
| If colorless, the reagent has expired; if blue, indicates phosphorus contamination. Prepare the Phosphorus Assay Reagent freshly before use. |
Notes:
(1) Reagent Ⅲ has a pungent odor, Reagent Ⅴ is toxic, and Reagent Ⅵ is corrosive. It is recommended to perform the experiment in a fume hood.
(2) It is best to use new beakers, glass rods, and glass pipettes for reagent preparation, or use disposable plasticware to avoid phosphorus contamination.
2. Sample Preparation
Notes:
(1) Fresh samples are recommended. If not used immediately for experimentation, samples can be stored at -80°C for 1 month. Processed samples must be tested on the same day.
(2) If protein concentration measurement is required, the use of Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
2.1 Plasma, Serum, and other liquid samples
Can be detected directly.
2.2 Animal tissue samples
Homogenize the tissue on ice with Extraction Buffer at a ratio of 0.1 g tissue to 1 mL Buffer. Centrifuge the homogenate at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.
2.3 Bacterial and cell samples
Collect bacteria or cells into a centrifuge tube, centrifuge at 4°C, 12,000 g for 1 min, and discard the supernatant. Add 1 mL of Extraction Buffer per 5×10⁶ bacteria or cells. Ultrasonicate to disrupt the bacteria or cells (power 20% or 200 W, ultrasonic for 3 s, interval 10 s, repeat 30 times). Centrifuge at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.
2.4 Plant samples
Rinse the plant sample with cold PBS to remove surface impurities. Homogenize the tissue on ice with Extraction Buffer at a ratio of 0.1 g tissue to 1 mL Buffer. Centrifuge the homogenate at 4°C, 8,000 g for 10 min. Collect the supernatant and keep it on ice for testing.
3. Experimental Steps
3.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 660 nm. For the visible spectrophotometer, zero it with deionized water.
3.2 Enzymatic Reaction (Add the following reagents to EP tubes)
Reagent (μL)
| Control Tube
| Test Tube
|
Assay Buffer
| 65
| 45 |
Working Reagent Ⅰ
| 60 | 60 |
Reagent Ⅱ
| 0 | 20 |
Sample
| 0 | 100 |
Mix thoroughly and place in a water bath at 37°C (for mammals) or 25°C (for other species) for exactly 10 minutes.
Working Reagent Ⅲ
| 25
| 25
|
Sample
| 100
| 0 |
Mix thoroughly, centrifuge at 4,000 g, room temperature for 10 minutes, and collect the supernatant.
3.3 Phosphorus Assay (Add the following reagents to a 96-well plate or micro glass cuvettes)
Reagent (μL)
| Blank Well
| Standard Well
| Control Well
| Test Well
|
0.5 μmol/mL Standard P Application Solution
| 0 | 20
| 0
| 0
|
Supernatant
| 0
| 0
| 20
| 20
|
Deionized water
| 20
| 0
| 0
| 0
|
Phosphorus Assay Reagent
| 200
| 200
| 200
| 200 |
3.4 Mix thoroughly and let stand at room temperature for 30 minutes. Measure the absorbance at 660 nm and record the values.
Note: A control well is required for each sample. Only one blank well and one standard well are needed. It is recommended to perform a preliminary experiment with 2-3 samples expected to have large differences before the formal experiment. If (ATest - AControl) is greater than 0.3, the sample can be further diluted with Extraction Buffer, and the calculated result should be multiplied by the dilution factor, or the amount of sample used for extraction can be reduced.
4. Calculation of Results
Note: We provide two types of calculation formulas: the derived process formula and the simplified formula. They are completely equivalent. It is recommended to use the simplified formula in bold as the final calculation formula.
4.1 Calculation of Na⁺/K⁺-ATPase Activity in Serum (Plasma) and other liquid samples
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per milliliter of serum (plasma).
Na⁺/K⁺-ATPase activity (U/mL) = CStandard × (ATest - AControl) ÷ (AStandard - ABlank) × VTotal ÷ VSample ÷ (T ÷ 60) = 7.5 × (ATest - AControl) ÷ (AStandard - ABlank)
4.2 Calculation of Na⁺/K⁺-ATPase Activity in Tissues, Bacteria, or Cells
(1) Calculation based on protein concentration
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per milligram of tissue protein.
Na⁺/K⁺-ATPase activity (U/mg) = CStandard × (ATest - AControl) ÷ (AStandard - ABlank) × VTotal ÷ (Cpr × VSample) ÷ (T ÷ 60) = 7.5 × (ATest - AControl) ÷ (AStandard - ABlank) ÷ Cpr
(2) Calculation based on sample fresh weight
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per gram of tissue.
Na⁺/K⁺-ATPase activity (U/g) = CStandard × (ATest - AControl) ÷ (AStandard - ABlank) × VTotal ÷ (VSample ÷ VSample Total × W) ÷ (T ÷ 60) = 7.5 × (ATest - AControl) ÷ (AStandard - ABlank) ÷ W
(3) Calculation based on bacterial or cell density
Definition: One unit of enzyme activity is defined as the amount of enzyme that hydrolyzes ATP to produce 1 μmol of inorganic phosphorus per hour per 10⁴ bacteria or cells.
Na⁺/K⁺-ATPase activity (U/10⁴) = CStandard × (ATest - AControl) ÷ (AStandard - ABlank) × VTotal ÷ (VSample ÷ VSample Total × 500) ÷ (T ÷ 60) = 0.015 × (ATest - AControl) ÷ (AStandard - ABlank)
Parameter Description:
CStandard: Concentration of the standard tube, 0.5 μmol/mL;
VTotal: Total volume of the enzymatic reaction, 0.25 mL;
VSample: Volume of sample added, 0.1 mL;
VSample Total: Volume of Extraction Buffer added, 1 mL;
T: Reaction time, 10 min;
Cpr: Sample protein concentration, mg/mL;
W: Sample fresh weight, g;
500: Total number of bacteria or cells, 5 million.
Precautions
1. Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.
2. If protein concentration measurement is required, the use of Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
3. Biochemical detection reagents are generally irritating, biologically toxic, etc. For your safety and health, please implement appropriate biosafety protection throughout the experiment, wear lab coats, masks, gloves, head covers, and other safety gear, and perform the experiment in a fume hood or biosafety cabinet.
4. This product is for scientific research use only. It is not intended for clinical diagnosis.
ZJ26F0333291
危险品化学品经营许可证（带存储）