计算溶液所需的质量、体积或浓度。
碱性磷酸酶检测试剂盒 (荧光法)
有货
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储存与运输
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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文献证明
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。
概述
碱性磷酸酶 (S-AKP/ALP)广泛分布于动物组织、土壤及微生物中，可催化磷酸单酯水解脱磷酸反应。生物样本中，血清或组织 AKP/ALP 活性异常可反映骨骼代谢、肝胆损伤、细胞分化状态，常用于病理模型评估与细胞成骨分化功能检测。土壤中碱性磷酸酶是关键土壤酶指标，参与土壤有机磷矿化分解，将有机磷转化为植物可利用无机磷，其活性高低直接表征土壤磷素供应能力、微生物代谢水平及土壤肥力状况，是土壤质量评价、施肥效应与生态修复研究的重要参数。
本试剂盒检测原理为碱性磷酸酶 (S-AKP/ALP) 能够在碱性环境中催化4-甲基伞形酮磷酸酯二钠盐(4-Methylumbelliferyl phosphate disodium salt, 4-MUP) 生成 4-甲基伞形酮 (4-Methylumbelliferone, 4-MU)和磷酸盐 (Phosphate)。其中，4-MU呈蓝色荧光，激发波长为360nm，发射波长为450nm。生成的4-MU荧光强度与碱性磷酸酶的活性成正比，通过检测4-MU的荧光强度最终计算出碱性磷酸酶的活性。
A1523089 | 组件 | 物理外观 | 100 T | 储存条件 | 单次测试使用量 |
A1523089A | 反应缓冲液 | 液体 | 10 mL | -20℃. | 78 µL |
A1523089B | 提取液 | 液体 | 20 mL | -20℃. | 按需 |
A1523089C | 4-MUP | 液体 | 0.2 mL | -20℃.避光 | 2 µL |
A1523089D | 4-MU (50mM) | 液体 | 0.2 mL | -20℃.避光 | 按需 |
A1523089E | 阳性对照 (200×) | 液体 | 20 µL | -20℃.避光 | 按需 |
使用方法
1. 试剂准备
(1) 将提取液，反应缓冲液，4-MUP和4-MU室温解冻，完全平衡至室温后再使用。
(2) 阳性对照冰上解冻。
2. 样品准备
(1) 组织样本：请尽量使用新鲜的组织样本进行测定。每10 mg组织样本加入100-200 μL提取液，快速冰上匀浆（匀浆器提前放冰上预冷）。匀浆后的样本，4℃，8,000 g 离心10 min，取上清液，置冰上待测。
(2) 血液、血浆或者尿液样品：对于血清和血浆样品，按照常规方法制备后，可直接用于该试剂盒的检测；尿液样本通常也可以直接用于该试剂盒检测。
(3) 细胞 (菌)样本：请尽量使用新鲜的细胞 (菌) 进行测定，而不要使用冻存的细胞 (菌) 进行测定。收集细胞 (菌)，首先用预冷的PBS清洗细胞 (菌) 2次 (PBS 重悬，4℃ 500 g 离心 5 min)，1× 10⁶ 个细胞 (菌)建议加入100-200 μL提取液重悬，冰浴超声波破碎 (200 W，超声 2 s，间隔 3 s，5 min)，4℃，8,000 g 离心 5 min，取上清液，置冰上待测。
3. 样本测定
(1) 标准曲线设置：取2 μL 4-MU (50 mM)标准品，加入398 μL 检测缓冲液，混匀，配制成400 μL浓度为250 μM的4-MU标准溶液，然后用检测缓冲液分别梯度稀释至125 μM，62.5 μM，31.25 μM，15.625 μM，7.8125 μM，3.906 μM和0 μM (空白孔)，然后各取20 μL不同浓度的标准溶液至96孔板当中，此时各孔4-MU物质的量分别为0、0.078、0.156、0.312、0.625、1.25、2.5、5 nmol。
(2) 阳性对照设置：取适量的阳性对照 (200×)，用反应缓冲液将其稀释200倍，然后取20 μL加入96孔板作为阳性样品。
(3) 样品测定：在96孔板中按照下表依次加入样本、标准品和阳性对照。
试剂名称 | 测定孔 | 空白孔 | 标准孔 | 阳性对照孔 |
待测样品 | 20 μL | - | - | - |
标准品 | - | - | 20 μL | - |
阳性对照 | - | - | - | 20 μL |
检测缓冲液 | 78 μL | 98 μL | 78 μL | 78 μL |
4-MUP | 2 μL | 2 μL | 2 μL | 2 μL |
混匀后于37 ℃反应20 min。反应结束后，读取荧光强度。ΔA=A测定 - A空白；Δ标准=A标准 - A空白。 |
4. 结果计算
(1) 标准曲线 ：Y=530.39*X + 54.044(仅供参考)；X是4-MU物质的量(nmol)，Y是△标准。算出样品ΔA的值，与标准曲线对比，即可算出样品中生成4-MU的量，然后换算成酶活单位。
(2) 酶活定义：一个酶活力单位 (Unit, U) 在25或37ºC，碱性的条件下，1分钟内可以生成1 μmol的4-MU。
注意事项
1. 4-MU和4-MUP等组分，尽量避免多次反复冻融且注意避光保存。
2. 如果样品活性高可缩短反应时间至10min以内，活性低则可延长至30min以上。
3. 本试剂盒仅用于科研用途，不得用于临床检测或其它用途。
4. 在实验过程中，请规范穿戴手套和实验服，避免皮肤直接接触试剂盒组分。
Alkaline phosphatase (S-AKP/ALP) is widely distributed in animal tissues, soil and microorganisms, and catalyzes the hydrolytic dephosphorylation of phosphate monoesters. In biological samples, abnormal AKP/ALP activity in serum or tissues reflects bone metabolism, hepatobiliary injury and cell differentiation status, and is frequently applied in pathological model evaluation and osteogenic differentiation assay. Soil-borne alkaline phosphatase serves as a critical soil enzyme indicator. It participates in the mineralization and decomposition of soil organic phosphorus, converting organic phosphorus into plant‑available inorganic phosphorus. Its activity directly characterizes soil phosphorus supply capacity, microbial metabolic level and soil fertility, acting as an essential parameter for soil quality assessment, fertilization‑effect investigation and ecological restoration research.
Alkaline phosphatase (S-AKP/ALP) catalyzes 4-Methylumbelliferyl phosphate disodium salt (4-MUP) to produce 4-Methylumbelliferone (4-MU) and phosphate under alkaline conditions. Among them, 4-MU exhibits blue fluorescence with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. The fluorescence intensity of the generated 4-MU is proportional to the activity of alkaline phosphatase. The activity of alkaline phosphatase can be finally calculated by measuring the fluorescence intensity of 4-MU.
A1523089 | Components | Appearance | 100T | Storage | volume per test |
A1523089A | Reaction Buffer | liquid | 10 mL | -20℃. | 78 µL |
A1523089B | Extraction Buffer | liquid | 20 mL | -20℃. | As need |
A1523089C | 4-MUP | liquid | 0.2 mL | -20℃.Store in the dark | 2 µL |
A1523089D | 4-MU (50mM) | liquid | 0.2 mL | -20℃.Store in the dark | As need |
A1523089E | Positive Control (200×) | liquid | 20 µL | -20℃.Store in the dark | As need |
Usage Method
1. Reagent Preparation
(1) Thaw Extraction Buffer, Reaction Buffer , 4-MUP and 4-MU at room temperature. Allow all reagents to fully equilibrate to room-temperature prior to use.
(2) Thaw Positive Control on ice.
2. Sample Preparation
(1) Tissue samples: Use fresh tissue samples for determination whenever possible. Add 100‑200 μL of Extraction Buffer per 10 mg tissue sample, and perform rapid homogenization on ice (pre‑cool the homogenizer on ice in advance). Centrifuge the homogenate at 8 000 g for 10 min at 4 °C. Collect the supernatant and keep it on ice for subsequent assay.
(2) Blood, plasma or urine samples: Serum and plasma samples can be directly assayed with this kit after routine preparation; urine samples are generally suitable for direct detection as well.
(3) Cell (bacterial) samples: Fresh cells/bacteria are recommended instead of frozen ones. Harvest cells/bacteria and wash twice with pre‑cooled PBS (resuspend in PBS, centrifuge at 500 g for 5 min at 4 °C). For 1×10⁶ cells or bacteria, resuspend in 100‑200 μL extraction solution. Lyse by sonication on ice (200 W, 2 s pulse on, 3 s pulse off, total 5 min). Centrifuge the lysate at 8 000 g for 5 min at 4 °C. Collect the supernatant and keep on ice for testing.
3. Sample Assay
(1) Standard curve preparation: Pipette 2 μL of 4-MU (50 mM) standard, add 398 μL Reaction Buffer, and mix well to prepare 400 μL of 250 μM 4-MU standard solution. Serially dilute this stock with Reaction Buffer to 125 μM, 62.5 μM, 31.25 μM, 15.625 μM, 7.8125 μM, 3.906 μM and 0 μM (blank well). Transfer 20 μL of each concentration of standard solution into separate wells of a 96-well plate. The corresponding amounts of 4-MU in each well are 0, 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, and 5 nmol, respectively.
(2) Positive control setup: Take an appropriate volume of positive control (200×), dilute it 200-fold with Reaction Buffer, then transfer 20 μL into the 96-well plate as the positive sample.
(3) Sample measurement: Add samples, standards and positive control sequentially into the 96-well plate according to the table below.
Reagent Name | Sample well | Blank well | Standard well | Positive well |
Sample to be tested | 20 μL | - | - | - |
Standard | - | - | 20 μL | - |
Positive Control | - | - | - | 20 μL |
Reaction Buffer | 78 μL | 98 μL | 78 μL | 78 μL |
4-MUP | 2 μL | 2 μL | 2 μL | 2 μL |
Mix thoroughly and incubate at 37 ℃ for 20 min. After the reaction, read the fluorescence intensity. ΔA = A<sub>sample</sub> − A<sub>blank</sub>; Δstandard = A<sub>standard</sub> − A<sub>blank</sub>. |
4. Calculation of Results
(1) Y = 530.39*X + 54.044 (for reference only); X represents the amount of 4-MU (nmol), and Y is Δstandard. Calculate the ΔA value of the sample. Compare it with the standard curve to obtain the amount of 4-MU produced in the sample, which is then converted into enzyme activity units.
(2) Enzyme activity definition: One unit (U) is defined as the amount of enzyme that produces 1 μmol of 4-MU per minute under alkaline conditions at 25 °C or 37 °C.
Precautions
1. Components such as 4-MU and 4-MUP should be protected from light and repeated freeze-thaw cycles as much as possible.
2. If the sample exhibits high enzyme activity, the reaction time can be shortened to less than 10 min; for samples with low activity, the reaction time may be extended to more than 30 min.
3. This kit is for research-only use. It shall not be applied for clinical diagnosis or any other purposes.
4. During the experiment, wear gloves and lab coats as standard laboratory practice to avoid direct skin contact with kit components.
规格
规格或纯度
BioReagent, 比色法, 适用于分析, 标曲法
英文名称
Alkaline Phosphatase Assay Kit (Fluorometric)
应用
细胞代谢
检测范围
0.025 U/L-12.5 U/L
检测方法
荧光法/吸光度法, 微量法
检测仪器
酶标仪
检测波长
Excitation wavelength 360 nm, emission wavelength 450 nm
敏感性
Light-sensitive
检测时长
20 min
样本体积
20 μL
储存与运输
储存条件
-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年
技术文档
高级数据
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