BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
碱性磷酸酶（Alkaline phosphatase，简称ALP或AKP）为一类磷酸酯酶，广泛分布于哺乳动物组织内，其活性所需最适pH 9.2~9.8。此酶主要存在于物质交换活跃之处（细胞膜），如肠上皮和肾近曲小管的刷状缘、附睾上皮之静纤毛、肝的毛细胆管膜以及微动脉和毛细血管动脉部之内皮，还见于内质网、高尔基复合体、吞饮小泡、肠上皮之溶酶体、中性粒细胞之中性颗粒以及平滑肌的细胞膜。
检测原理：Para-nitrophenyl phosphate (pNPP)为一种常用的磷酸酶显色底物，在酸性条件下，可在碱性磷酸酶的作用下生成p-nitrophenol。在碱性条件下p-nitrophenol转变成醌式结构，呈较深的黄色，产物黄色越深说明碱性磷酸酶活性越高，反之则酶活性越低，通过分光光度比色法测定400～415nm处吸光度，据此通过比色分析就可以计算出碱性磷酸酶活性水平。
适用样本：细胞或组织的裂解液或匀浆液、血浆、血清、尿液等
| A1509256 | Component | 60T | Storage |
| A1509256A | ALP Assay Buffer | 50 mL | 2-8℃ |
| A1509256B | pNPP | 2×1EA | -20℃. Store in the dark. |
| A1509256C | p-nitrophenol (10mM) | 0.2 mL | -20℃. Store in the dark. |
| A1509256D | Stopping Solution | 75 mL | RT. |
自备仪器和试剂
操作步骤（仅供参考）
1、样品准备
1.1 细胞或组织样品
取恰当细胞或组织裂解液，如果有必要可用PBS或生理盐水进行适当匀浆，一般细胞数量在10⁶以上，组织应在100mg以上，3000-4000g离心取上清，-20℃冻存，用于碱性磷酸酶的检测。
1.2 血浆、血清和尿液样品
血浆、血清按照常规方法制备后可以直接用于该试剂盒的测定，尿液通常也可以直接用于测定，-20℃冻存，但为了消除样品本身颜色的干扰，需设置加了血浆或血清但不加底物的对照。
1.3 高活性样品
如果样品中含有较高活性的碱性磷酸酶，可以使用原有的裂解液或PBS等进行稀释，也可以采用ALP Assay Buffer稀释。
2、配制显色工作液
取出1支pNPP，恢复至室温后溶解于15mL ALP Assay Buffer，混匀，冰上预冷备用，新配制的显色工作液应在6h内用完。
3、配制标准品工作液
取出 p-nitrophenol (10mM) 恢复至室温后，取0.1mL溶解于1.9mL ALP Assay Buffer，使浓度达到0.5mM即获得p-nitrophenol (0.5mM)，该试剂-20℃保存2周有效。用p-nitrophenol (0.5mM) 按下表继续稀释标准品：
加入物（mL） | 1 | 2 | 3 | 4 | 5 | 6 |
ALP Assay Buffer | 0.45 | 0.4 | 0.3 | 0.25 | 0.1 | 0 |
p-nitrophenol (0.5mM) | 0.05 | 0.1 | 0.2 | 0.25 | 0.4 | 0.5 |
p-nitrophenol浓度 (μM) | 50 | 100 | 200 | 250 | 400 | 500 |
4、ALP加样
按照下表设置空白对照管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡。如果样品中的碱性磷酸酶活性过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置平行管。
加入物（mL） | 对照管 | 标准管 | 测定管 |
ALP Assay Buffer | 0.5 | — | — |
标准品工作液（1-6号） | — | 0.5 | — |
待测样品 | — | — | 0.5 |
显色工作液 | 0.5 | 0.5 | 0.5 |
混匀，37℃孵育20min。
Stopping Solution | 1 | 1 | 1 |
5、ALP测定
以对照调零，比色杯光径1cm，分光光度计测定410nm处吸光度，如果无法检测410nm，亦可检测400～415nm范围内吸光度，一般15min内检测完毕。
6、结果计算
碱性磷酸酶活性单位的定义：在pH9.8的缓冲液中，37℃条件下，每分钟水解para-nitrophenyl phosphate显色底物产生1微摩尔p-nitrophenol所需的碱性磷酸酶的量定义为一个酶活力单位。
加入0.5mL的1号标准管物质时，其酶活力单位为50uM/20min=2.5U/L，依此类推，加入浓度分别为100μM、200μM、250μM、400μM、500μM的p-nitrophenol，活力依次为5、10、12.5、20、25U/L。以酶活力为横坐标，以对应的吸光度为纵坐标绘制标准曲线，根据酶活性定义，计算出样品中的碱性磷酸酶活性。
注意事项
1、待测样品中不能含有磷酸酶抑制剂，同时需避免反复冻融。
2、建议每次测定时都做标准曲线，以使标准更准确，另外标准品需避免反复冻融。
3、所测样本的值高于标准曲线的上限，应用ALP Assay Buffer稀释样品后重新测定。
4、1支显色工作液配制后需当日使用完毕，因此请注意适当多准备一些样品一起检测。
5、p-nitrophenol溶液对人体有害，反应终止液有腐蚀性，请小心操作。
6、如果希望进行酶活性的绝对定量，进行酶反应时应精确计时，此时推荐采用孵育30min或更长时间，以减小操作过程中的时间误差。
7、待测样品中碱性磷酸酶活性较低时，可适当延长孵育时间至30min。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
9、试剂开封后请尽快使用，以防影响后续实验效果。
Alkaline phosphatase (ALP or AKP) is a type of phosphatase widely distributed in mammalian tissues, with an optimal pH range of 9.2–9.8 for its activity. This enzyme is primarily located in areas of active substance exchange (cell membranes), such as the brush borders of intestinal epithelium and renal proximal tubules, the stereocilia of epididymal epithelium, the canalicular membranes of the liver, and the endothelium of arterioles and arterial capillaries. It is also found in the endoplasmic reticulum, Golgi apparatus, pinocytotic vesicles, lysosomes of intestinal epithelium, specific granules of neutrophils, and the cell membranes of smooth muscle.
Detection Principle
Para-nitrophenyl phosphate (pNPP) is a commonly used chromogenic substrate for phosphatases. Under alkaline conditions, it is hydrolyzed by alkaline phosphatase to produce p-nitrophenol. In an alkaline environment, p-nitrophenol converts to a quinoid structure, exhibiting a deep yellow color. The deeper the yellow color of the product, the higher the alkaline phosphatase activity, and vice versa. The absorbance is measured at 400–415 nm using spectrophotometry, allowing the calculation of alkaline phosphatase activity levels through colorimetric analysis.
Applicable Samples: Cell or tissue lysates or homogenates, plasma, serum, urine, etc.
| A1509256 | Component | 60T | Storage |
| A1509256A | ALP Assay Buffer | 50 mL | 2-8℃ |
| A1509256B | pNPP | 2×1EA | -20℃. Store in the dark. |
| A1509256C | p-nitrophenol (10mM) | 0.2 mL | -20℃. Store in the dark. |
| A1509256D | Stopping Solution | 75 mL | RT. |
Reagents, consumables and Equipments not provided
Operating Steps (for reference only)
1. Sample Preparation
1.1 Cell or Tissue Samples
Take an appropriate amount of cell or tissue lysate. If necessary, homogenize appropriately with PBS or physiological saline. Generally, use cell counts above 10⁶ or tissue amounts above 100 mg. Centrifuge at 3000–4000 g and collect the supernatant. Store at -20°C for alkaline phosphatase detection.
1.2 Plasma, Serum, and Urine Samples
Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit. Urine can also typically be used directly. Store at -20°C. To eliminate interference from the sample's own color, set up a control containing plasma or serum but without the substrate.
1.3 High-Activity Samples
If the sample contains high alkaline phosphatase activity, dilute it with the original lysis buffer, PBS, or ALP Assay Buffer.
2. Preparation of Chromogenic Working Solution
Take one tube of pNPP, allow it to reach room temperature, and dissolve it in 15 mL of ALP Assay Buffer. Mix well and pre-cool on ice. The freshly prepared chromogenic working solution should be used within 6 hours.
3. Preparation of Standard Working Solution
Take the p-nitrophenol (10 mM), allow it to reach room temperature, and mix 0.1 mL with 1.9 mL of ALP Assay Buffer to obtain a concentration of 0.5 mM (p-nitrophenol, 0.5 mM). Store at -20°C. This solution is stable for 2 weeks. Further dilute the standard using p-nitrophenol (0.5 mM) according to the table below:
Tube Number | 1 | 2 | 3 | 4 | 5 | 6 |
ALP Assay Buffer (mL) | 0.45 | 0.4 | 0.3 | 0.25 | 0.1 | 0 |
p-nitrophenol (0.5 mM) (mL) | 0.05 | 0.1 | 0.2 | 0.25 | 0.4 | 0.5 |
| p-nitrophenol Concentration (μM) | 50 | 100 | 200 | 250 | 400 | 500 |
4. ALP Sample Addition
Set up blank control, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the alkaline phosphatase activity in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.
Substance Added (mL) | Control Tube | Standard Tube | Test Tube |
ALP Assay Buffer | 0.5 | — | — |
Standard Working Solution (Tubes 1-6) | — | 0.5 | — |
Sample | — | — | 0.5 |
Chromogenic Working Solution | 0.5 | 0.5 | 0.5 |
Mix well and incubate at 37°C for 20 minutes.
Stopping Solution | 1 | 1 | 1 |
5. ALP Measurement
Zero the spectrophotometer with the control tube. Using a 1 cm light path cuvette, measure the absorbance at 410 nm. If 410 nm is not available, absorbance within the 400–415 nm range can be measured. Detection should generally be completed within 15 minutes.
6. Result Calculation
Definition of Alkaline Phosphatase Activity Unit: One unit of enzyme activity is defined as the amount of alkaline phosphatase required to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute under pH 9.8 buffer conditions at 37°C.
When 0.5 mL of the standard from Tube 1 is added, its enzyme activity is 50 μM / 20 min = 2.5 U/L. Similarly, for p-nitrophenol concentrations of 100 μM, 200 μM, 250 μM, 400 μM, and 500 μM, the corresponding activities are 5, 10, 12.5, 20, and 25 U/L, respectively. Plot a standard curve with enzyme activity as the x-axis and the corresponding absorbance as the y-axis. Calculate the alkaline phosphatase activity in the sample based on the definition of enzyme activity.
Precautions
The test sample must not contain phosphatase inhibitors, and repeated freeze-thaw cycles should be avoided.
It is recommended to prepare a fresh standard curve for each assay to ensure accuracy. Also, avoid repeated freeze-thaw cycles of the standard.
If the measured sample value exceeds the upper limit of the standard curve, dilute the sample with ALP Assay Buffer and repeat the measurement.
One prepared batch of chromogenic working solution must be used on the same day. Therefore, prepare an appropriate number of samples for simultaneous detection.
p-nitrophenol solution is harmful to humans, and the stopping solution is corrosive. Handle with care.
For absolute quantification of enzyme activity, precise timing of the enzymatic reaction is necessary. In this case, incubating for 30 minutes or longer is recommended to minimize time errors during operation.
If the alkaline phosphatase activity in the sample is low, the incubation time can be appropriately extended to 30 minutes.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
| A1509256 | Component | 60T | Storage |
| A1509256A | ALP Assay Buffer | 50 mL | 2-8℃ |
| A1509256B | pNPP | 2×1EA | -20℃. Store in the dark. |
| A1509256C | p-nitrophenol (10mM) | 0.2 mL | -20℃. Store in the dark. |
| A1509256D | Stopping Solution | 75 mL | RT. |
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