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。
乙酰胆碱酯酶（AchE）是一种主要存在于神经系统中的丝氨酸水解酶，以多种变体形式广泛存在于各种动物组织和血清中，可催化神经递质乙酰胆碱水解，从而终止胆碱神经信号的传递，在神经传导调节过程中起重要作用，同时还具有诱导轴突生长和突触形成，促进造血细胞形成等功能。
检测原理：AchE催化Ach水解生成胆碱，胆碱与二硫对硝基苯甲酸（DTNB）作用生成5-巯基-硝基苯甲酸（TNB）；TNB在412nm处有吸收峰，通过测定412nm吸光度增加速率，计算AchE活性。
适用样本：新鲜血清（浆）、动物组织、神经细胞
| A1506759 | Component | 96T | Storage |
| A1506759A | Extraction Buffer | 120 mL | 2-8℃ |
| A1506759B | Assay Buffer | 20 mL | 2-8℃ |
| A1506759C | Chromogen | 1.3 mL | 2-8℃. Store in the dark. |
| A1506759D | Substrate | 1.3 mL | 2-8℃. Store in the dark. |
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
1、酶标仪或可见光分光光度计（能测412nm处的吸光度）
2、恒温箱、制冰机、低温离心机
3、96孔板或微量玻璃比色皿、可调节式移液枪及枪头
4、去离子水
5、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 |
| Chromogen | 即用型；使用前平衡到室温 | 4℃避光保存；有刺激性，建议在使用过程中做好个人防护。 |
| Substrate | 即用型；使用前平衡到室温 | 4℃避光保存 |
| 工作液 | 将Assay Buffer，Chromogen和Substrate按照16:1:1的比例混合均匀 | 临用前配制，现配现用 |
2、样本制备
2.1 动物组织
称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8,000g，4℃离心10min，取上清液，置冰上待测。
2.2 神经细胞
收集500万细胞到离心管内，用冷PBS清洗细胞，离心后弃上清，加入1mL Extraction Buffer，冰浴超声波破碎细胞5min（功率20%或200W，超声3s，间隔7s，重复30次），然后8,000g，4℃离心10min，取上清液，置冰上待测。
2.3 新鲜血清（浆）等液体样本
直接测定。
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。
②、对于脂肪含量较高的动物组织，离心后移除上层脂肪，再取上清液。
③、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 酶标仪或可见分光光度计预热30min以上，调节波长到412nm，可见分光光度计用去离子水调零。
3.2 恒温箱预热到37℃，工作液置于恒温箱中预热30min。
3.3 在96孔板或微量玻璃比色皿中依次加入20μL 样本，180μL 工作液，迅速混匀，37℃孵育3min，于412nm处测定3min内吸光值变化。第20s吸光值记为A1，第200s吸光值记为
注意：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ΔA小于0.002可适当加大样本量。如果ΔA大于0.8，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 使用96孔板测定的计算公式
(1) 动物组织AchE活性
A. 按照蛋白浓度计算
活性单位定义：每毫克蛋白每分钟催化产生1nmol TNB的酶量为1个酶活单位。
4.2 使用微量玻璃比色皿进行测定的计算公式
将上述计算公式中光径d: 0.5cm调整为d: 1cm进行计算即可。
5、结果展示
图1. 试剂盒测定小鼠脑、小鼠肝脏中的AchE活性
注意事项
本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Acetylcholinesterase (AChE) is a serine hydrolase primarily found in the nervous system. It exists widely in various animal tissues and serum in multiple isoforms. AChE catalyzes the hydrolysis of the neurotransmitter acetylcholine, thereby terminating cholinergic nerve signal transmission. It plays a crucial role in the regulation of neural conduction, and also functions in inducing axon growth, promoting synapse formation, and facilitating hematopoietic cell formation.
Detection Principle
AChE catalyzes the hydrolysis of acetylcholine (Ach) to produce choline. Choline reacts with 5,5'-Dithio-bis-(2-nitrobenzoic acid) (DTNB) to generate 5-thio-2-nitrobenzoic acid (TNB). TNB has an absorption peak at 412 nm. The AChE activity is calculated by determining the rate of increase in absorbance at 412 nm.
Applicable Samples: Fresh serum (plasma), animal tissues, nerve cells.
| A1506759 | Component | 96T | Storage |
| A1506759A | Extraction Buffer | 120 mL | 2-8℃ |
| A1506759B | Assay Buffer | 20 mL | 2-8℃ |
| A1506759C | Chromogen | 1.3 mL | 2-8℃. Store in the dark. |
| A1506759D | Substrate | 1.3 mL | 2-8℃. Store in the dark. |
Note: Before formal testing, it is recommended to perform a preliminary assay using 2-3 samples expected to have significant differences.
Materials Required but Not Provided
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 412 nm)
Incubator, ice maker, refrigerated centrifuge
96-well plate or micro glass cuvettes, adjustable pipettes, and tips
Deionized water
Homogenizer (if processing tissue samples)
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Chromogen | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. Irritant. Wear appropriate personal protective equipment. |
| Substrate | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Working Solution | Mix Assay Buffer, Chromogen, and Substrate uniformly in a 16:1:1 ratio. | Prepare immediately before use. |
2. Sample Preparation
2.1 Animal Tissue
Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize in an ice bath. Centrifuge at 8,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.
2.2 Nerve Cells
Collect 5 million cells into a centrifuge tube. Wash the cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt the cells using an ultrasonic cell disruptor in an ice bath for 5 minutes (20% power or 200 W, ultrasound for 3 s, interval for 7 s, repeat 30 times). Then centrifuge at 8,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.
2.3 Fresh Serum (Plasma) and Other Liquid Samples
Assay directly.
Notes:
① Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.
② For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.
③ If measuring protein concentration, the Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
3. Assay Procedure
3.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 412 nm. Zero the visible spectrophotometer with deionized water.
3.2 Preheat the incubator to 37°C. Pre-warm the Working Solution in the incubator for 30 minutes.
3.3 Add 20 μL of sample and 180 μL of pre-warmed Working Solution into a 96-well plate or micro glass cuvette. Mix rapidly and incubate at 37°C for 3 minutes. Monitor the absorbance change at 412 nm over 3 minutes. Record the absorbance at 20 seconds as A1 and the absorbance at 200 seconds as A2. Calculate ΔA = A2 - A1.
Note: Preliminary tests with 2-3 samples expected to have significant differences are recommended. If ΔA is less than 0.002, consider increasing the sample volume appropriately. If ΔA is greater than 0.8, the sample can be further diluted with Extraction Buffer (multiply the result by the dilution factor) or the sample amount used for extraction can be reduced.
4. Calculation of Results
Note: We provide both a detailed derivation formula and a simplified formula. They are exactly equivalent. The simplified formula (in bold) is recommended as the final calculation formula.
4.1 Calculation Formulas for 96-Well Plate Assay
(1) AChE Activity in Animal Tissue
A. Calculation based on protein concentration
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per mg of protein.
AChE Activity (U/mg prot)
= (ΔA ÷ ε ÷ d × Vreaction total × 10⁹) ÷ (Cpr × Vsample) ÷ T
= 490 × ΔA ÷ Cpr × n B. Calculation based on sample weight
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per gram of tissue. AChE Activity (U/g fresh weight)
= (ΔA ÷ ε ÷ d × Vreaction total × 10⁹) ÷ (W × Vsample ÷ Vsample total) ÷ T
= 490 × ΔA ÷ W × n
(2) AChE Activity in Nerve Cells
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per 10⁴ cells. AChE Activity (U/10⁴ cells)
= (ΔA ÷ ε ÷ d × Vreaction total × 10⁹) ÷ (Cell Number × Vsample ÷ Vsample total) ÷ T
= 490 × ΔA ÷ 500 × n = 0.98 × ΔA × n
(3) AChE Activity in Serum (Plasma) and Other Liquid Samples
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 nmol of TNB per minute per mL of sample. AChE Activity (U/mL)
= (ΔA ÷ ε ÷ d × Vreaction total × 10⁹) ÷ Vsample ÷ T
= 490 × ΔA × n
Parameter Description:
ε: Molar extinction coefficient of TNB, 13.6 × 10³ L/mol/cm
d: Light path for the 96-well plate, 0.5 cm
Vreaction total: Total reaction volume, L;
200 μL = 2 × 10⁻⁴ L
10⁹: Conversion factor from moles to nmoles;
1 mol = 1 × 10⁹ nmol
Cpr: Protein concentration, mg/mL
Vsample: Volume of supernatant added, 0.02 mL
T: Reaction time, 3 min
n: Sample dilution factor
W: Sample weight, g
Vsample total: Volume of extraction buffer, 1 mL
500: Total cell number, 5 million (500 × 10⁴)
4.2 Calculation Formulas for Micro Glass Cuvette Assay
Adjust the light path d in the formulas above from 0.5 cm to 1 cm and calculate accordingly.
Precautions
This product is for research use only. Not for use in diagnostic procedures. For your safety and health, please wear lab coats and disposable gloves during operation.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131308 |
分析证书 | A1506759 |
危险品化学品经营许可证（带存储）