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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（AKP/ALP）是一种能够将对应底物去磷酸化的含锌糖蛋白酶，广泛分布于人体各脏器中，在碱性 条件下，可以催化多种底物的水解，包括各种天然及人工合成的磷脂单酯化合物，通过水解磷酸单酯将底物分子上的磷酸基团除去，生成磷酸根离子和自由羟基。碱性磷酸酶可作为免疫诊断试剂的标记酶，并在骨骼、肝胆系统疾病的诊断和鉴别方面具有重要应用。
检测原理：AKP/ALP在碱性环境中催化磷酸苯二钠生成游离酚，酚与4-氨基安替比林和铁氰化钾反应生成红色亚醌衍生物，产物在510nm处具有特征吸收峰，通过吸光值变化即可表征AKP/ALP的活性。
适用样本：血清（浆）、动物组织、尿液
| A1492732 | Component | 48T | 96T | Storage |
| A1492732A | Extraction Buffer | 60 mL | 120 mL | 2-8℃ |
| A1492732B | Chromogen A | 2.5 mL | 5 mL | 2-8℃. Store in the dark. |
| A1492732C | Chromogen B | 2.5 mL | 5 mL | 2-8℃. Store in the dark. |
| A1492732D | Chromogen C | 7.5 mL | 15 mL | 2-8℃. Store in the dark. |
| A1492732E | Standard | 0.5 mL | 0.5 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510nm处的吸光度）
恒温箱、制冰机、低温离心机
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 |
| Chromogen A | 即用型；使用前平衡到室温 | 4℃避光保存；有毒，建议在通风橱进行实验 |
| Chromogen B | 即用型；使用前平衡到室温 | 4℃避光保存 |
| Chromogen C | 即用型；使用前平衡到室温 | 4℃避光保存；有毒，建议在通风橱进行实验 |
| Standard | 即用型；使用前平衡到室温 | 4℃保存；有刺激性气味，建议在通风橱进行实验 |
2、样本制备
注意：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。
2.1 组织样本：
称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，10,000rpm，4℃离心10min，取上清液，置冰上待测。
2.2 血清（浆）：血浆和血清可以直接用于测定。血浆制备时不能用EDTA和柠檬酸盐，可用其它抗凝剂。
2.3 尿液：直接测定。
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、实验步骤
3.1 酶标仪或可见光分光光度计预热30min以上，调节波长到510nm，可见光分光光度计用去离子水调零。
3.2 按照下表进行加样及反应：
| 试剂 | 空白孔 (μL) | 标准孔 (μL) | 对照孔 (μL) | 测定孔 (μL) |
| 去离子水 | 4 | 0 | 0 | 0 |
| Standard | 0 | 4 | 0 | 0 |
| Sample | 0 | 0 | 0 | 4 |
| Chromogen A | 40 | 40 | 40 | 40 |
| Chromogen B | 40 | 40 | 40 | 40 |
混匀后置于37℃孵育15min
| Chromogen C | 120 | 120 | 120 | 120 |
| Sample | 0 | 0 | 4 | 0 |
混匀后于510nm测定吸光度，其中标准和空白只需做一个，每个样本均需设置对照，各孔的吸光度分别记为A空白 ，A标准 ，A对照 ，A测定 。
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 按蛋白浓度计算
活性单位定义：37℃中每毫克蛋白每分钟催化产生1μmol酚为一个酶活力单位。
AKP/ALP (U/mgprot)=[C标准品 ×(A测定 -A对照 )÷(A标准 -A空白 )×V样 ]÷(Cpr×V样 )÷T=0.133×(A测定 -A对照 )÷(A标准 -A空白 )÷Cpr
4.2 按样本鲜重计算
活性单位定义：37℃中每克组织每分钟催化产生1μmol酚为一个酶活力单位。
AKP/ALP (U/g鲜重)=[C标准品 ×(A测定 -A对照 )÷(A标准 -A空白 )×V样 ]÷(W÷V提取 ×V样 )÷T=0.133×(A测定 -A对照 )÷(A标准 -A空白 )÷W
4.3 按液体体积计算
活性单位定义：37℃中每毫升血液或尿液每分钟催化产生1μmol酚为一个酶活力单位。
AKP/ALP (U/mL)=[C标准品 ×(A测定 -A对照 )÷(A标准 -A空白 )×V样 ]÷V 样 ÷T=0.133×(A测定 -A对照 )÷(A标准 -A空白 )
参数说明
C标准品 ：标准品的浓度，2μmol/mL；
V样 ：加入反应体系中上清液体积，0.004mL；
T：反应时间，15min；
V提取 ：加入提取液体积，1 mL；
W：样本鲜重，g；
Cpr：上清液蛋白质浓度，mg/mL
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、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。
3、本产品仅供科学研究使用，不适用于临床诊断。
Alkaline Phosphatase (AKP/ALP) is a zinc-containing glycoprotein enzyme capable of dephosphorylating corresponding substrates. It is widely distributed in various human organs. Under alkaline conditions, it can catalyze the hydrolysis of various substrates, including natural and synthetic phosphate monoester compounds, removing phosphate groups from substrate molecules via hydrolysis of phosphate monoesters to generate phosphate ions and free hydroxyl groups. Alkaline phosphatase can serve as a label enzyme for immunodiagnostic reagents and has important applications in the diagnosis and differentiation of skeletal and hepatobiliary system diseases.
Detection Principle: In an alkaline environment, AKP/ALP catalyzes the hydrolysis of disodium phenyl phosphate to generate free phenol. The phenol then reacts with 4-aminoantipyrine and potassium ferricyanide to form a red quinone derivative. This product has a characteristic absorption peak at 510 nm. The change in absorbance is used to characterize AKP/ALP activity.
Applicable Samples: Serum (plasma), animal tissues, urine.
| A1492732 | Component | 48T | 96T | Storage |
| A1492732A | Extraction Buffer | 60 mL | 120 mL | 2-8℃ |
| A1492732B | Chromogen A | 2.5 mL | 5 mL | 2-8℃. Store in the dark. |
| A1492732C | Chromogen B | 2.5 mL | 5 mL | 2-8℃. Store in the dark. |
| A1492732D | Chromogen C | 7.5 mL | 15 mL | 2-8℃. Store in the dark. |
| A1492732E | Standard | 0.5 mL | 0.5 mL | 2-8℃ |
Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
User-Provided Instruments and Reagents
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 510 nm)
Incubator, Ice maker, Low-temperature centrifuge
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 | Precautions |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Chromogen A | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. Toxic; recommended to handle in a fume hood. |
| Chromogen B | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Chromogen C | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. Toxic; recommended to handle in a fume hood. |
| Standard | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. Irritating smell; recommended to handle in a fume hood. |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.
2.1 Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 10,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.2 Serum (Plasma): Plasma and serum can be used directly for assay. EDTA and citrate should not be used as anticoagulants for plasma preparation; other anticoagulants are acceptable.
2.3 Urine: Detect directly.
For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. Assay Steps
3.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 510 nm. For spectrophotometers, zero the instrument with deionized water.
3.2 Assay Procedure: Add reagents and perform the reaction according to the table below:
| Reagent | Blank Well (µL) | Standard Well (µL) | Control Well (µL) | Test Well (µL) |
| Deionized Water | 4 | 0 | 0 | 0 |
| Standard | 0 | 4 | 0 | 0 |
| Sample | 0 | 0 | 0 | 4 |
| Chromogen A | 40 | 40 | 40 | 40 |
| Chromogen B | 40 | 40 | 40 | 40 |
Mix well and incubate at 37°C for 15 minutes.
| Chromogen C | 120 | 120 | 120 | 120 |
| Sample | 0 | 0 | 4 | 0 |
Mix well and measure the absorbance at 510 nm.
Note: The Standard and Blank wells need to be set up only once. Each sample requires a corresponding Control well.
Record the absorbances as A blank , A standard , A control , and A test .
4. Result Calculation
Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas.
4.1 Based on Protein Concentration:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per mg of protein at 37°C.**
Formula:
AKP/ALP (U/mg prot) = [Cstandard × (Atest - Acontrol ) ÷ (Astandard - Ablank ) × Vsample ] ÷ (Cpr × Vsample ) ÷ T = 0.133 × (Atest - Acontrol ) ÷ (A standard - Ablank ) ÷ Cpr
4.2 Based on Sample Fresh Weight:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per gram of fresh tissue at 37°C.**
Formula:
AKP/ALP (U/g fresh weight) = [Cstandard × (Atest - Acontrol ) ÷ (Astandard - Ablank ) × Vsample ] ÷ (W ÷ Vextract × Vsample ) ÷ T = 0.133 × (Atest - Acontrol ) ÷ (Astandard - Ablank ) ÷ W
4.3 Based on Liquid Volume:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per mL of blood or urine at 37°C.**
Formula:
AKP/ALP (U/mL) = [Cstandard × (Atest - Acontrol ) ÷ (Astandard - Ablank ) × Vsample ] ÷ Vsample ÷ T = 0.133 × (Atest - Acontrol ) ÷ (Astandard - A blank )
Parameter Description:
Cstandard : Concentration of the standard, 2 μmol/mL
Vsample : Volume of supernatant added to the reaction system, 0.004 mL
T: Reaction time, 15 min
Vextract : Volume of Extraction Buffer added, 1 mL
W: Sample fresh weight, g
Cpr: Protein concentration of the supernatant, mg/mL
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737423 |
分析证书 | A1492732 |
| ZJ25F1029028 |
分析证书 | A1492732 |
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