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天门冬氨酸氨基转移酶（AST, 旧称谷草转氨酶 GOT, EC 2.6.1.1）广泛分布于动植物组织，主要存在于肝脏、心肌、骨骼肌、肾脏，当组织细胞发生损伤破裂时，酶大量释放至体液中，因此 AST 活力是评价组织细胞损伤程度的重要生化指标。
本试剂盒检测原理是样本中 AST 催化 L-天冬氨酸与α-酮戊二酸发生氨基转移，生成草酰乙酸与 L-谷氨酸。苹果酸脱氢酶（MDH）将生成的草酰乙酸还原为 L-苹果酸，同时消耗 NADH 生成 NAD⁺。NADH 在 340 nm 具有强紫外特征吸收，NAD⁺在此波长几乎无吸收，测定 340 nm 处吸光度下降速率即可计算 AST 活性。
组分与储存：
* 请在实验前检查各组分的量。 * 各组分在规格基础上额外提供 10% 的量用于进行标准曲线制作或预实验 |
自备仪器与试剂：
| 自备资源类型 | 资源名称 | 备注 |
|---|---|---|
| 仪器 | 酶标仪 | 能测 340 nm 处的吸光度 |
| 耗材 | 96 孔酶标板 | UV 板 |
| 试剂 | PBS | 提取/稀释样本用 |
| 其他 | 匀浆器（组织）、超声破碎仪（细胞或细菌）、恒温箱、低温离心机、可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
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. 对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁货号B665595的BCA蛋白定量试剂盒。
3. 本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4. 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。
5. 本产品仅供科学研究使用，不适用于临床诊断。
实验流程：
1. 试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
|---|---|---|
| Reagent Ⅰ | 即用型；使用前平衡到室温 | 2-8℃ 保存 |
| Reagent Ⅱ | 即用型；使用前低速离心，平衡到室温 | 2-8℃ 保存 |
| Reagent Ⅲ | 即用型；使用前平衡到室温 | 2-8℃ 保存 |
2. 样本制备
注意：推荐使用新鲜样本。样本尽量新鲜提取，如果不能当天提取，则把完整细胞或者分装的组织块保存在-80℃，一个月内使用。当准备好进行该项实验时，将样本从-80℃拿出后，冰上解冻。但是请注意，这可能会影响样本的稳定性，实验结果可能会低于预期。
2.1 动植物组织：称取约 0.1 g 组织样本，加入 1 mL PBS，冰浴匀浆，12000 g，4℃离心 10 min，取上清液，置冰上待测。
2.2 细胞或细菌：收集 5×10⁶ 个细胞或细菌，用冷 PBS 清洗细胞或细菌，800 g 离心 2 min，弃上清液。加入 1 mL PBS，冰浴超声波破碎细胞或细菌 5 min（功率 20%或 200 W，超声 3 s，间隔 7 s，重复 30 次），12000 g，4℃离心 10 min，取上清液，置冰上待测。
2.3 血清（浆）等液体样本：直接检测。
3. 实验步骤
3.1 酶标仪准备：预热 30 min 以上，调节波长到 340 nm。
3.2 Working Solution 的配制：按照每孔 200 μL（Reagent Ⅰ 200 μL：Reagent Ⅱ 0.2 μL，配制体积比 1000：1）配制 Working Solution 并混合均匀，需现用现配。
3.3 检测体系配制：在 96 孔板中按照如下方式操作：
| 试剂 | 测定孔（μL） |
|---|---|
| 样本 | 20 |
| Working Solution | 200 |
| 混匀，37℃孵育 5 min | |
| Reagent Ⅲ | 80 |
3.4 吸光度检测：混匀，记录 340 nm 处 37℃孵育 5 min 的吸光值 A₁ 和 10 min 的吸光值 A₂。
结果计算：
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
1. 数据处理
计算ΔA=A₁-A₂。
2. AST 活力的计算
2.1 按蛋白浓度计算：
酶活定义：在 37℃条件下，每 g 组织蛋白在反应体系中每分钟消耗 1 μmol NADH 定义为一个酶活性单位。
AST (U/g prot)=[ΔA÷(ε×d)×10⁶×V反总]÷(V样×Cpr)÷T=535.9×ΔA÷Cpr
2.2 按样本鲜重计算：
酶活定义：在 37℃条件下，每 g 组织在反应体系中每分钟消耗 1 μmol NADH 定义为一个酶活性单位。
AST (U/g)=[ΔA÷(ε×d)×10⁶×V反总]÷(W×V样÷V样总)÷T=0.5359×ΔA÷W
2.3 按细胞或细菌数量计算：
酶活定义：在 37℃条件下，每 10⁶ 个细胞或细菌在反应体系中每分钟消耗 1 μmol NADH 定义为一个酶活性单位。
AST (U/10⁶)=[ΔA÷(ε×d)×10⁶×V反总]÷(N×V样÷V样总)÷T=0.5359×ΔA÷N
2.4 按液体体积计算：
酶活定义：在 37℃条件下，每 L 液体样本在反应体系中每分钟消耗 1 μmol NADH 定义为一个酶活性单位。
AST (U/L)=[ΔA÷(ε×d)×10⁶×V反总]÷V样÷T=535.9×ΔA ε：NADH 摩尔消光系数，6.22×10³ L/mol/cm；d：96 孔 UV 板光径，0.9 cm；10⁶：单位换算系数，1 mol=10⁶ μmol；V反总：反应体系总体积，3×10⁻⁴ L；V样：加入反应体系中样本体积，2×10⁻⁵ L；Cpr：样本蛋白质浓度，g/L；T：反应时间，5 min；V样总：加入 PBS 体积，10⁻³ L；W：样本质量，g；N：细胞数量，以 10⁶ 为单位（例如细胞数量为 5×10⁶，N=5）。
结果展示：
示例-1：取 0.1 g 小鼠心脏，按上述步骤操作，用 PBS 稀释 90 倍后，用 96 孔 UV 板测得ΔA=A₁-A₂=2.190-2.007=0.183，按样本鲜重计算含量得：稀释 90 倍的小鼠心脏 AST (U/g)=0.5359×ΔA÷W=0.981 U/g。
常见问题：
检测得到的样本ΔA 过高或者过低怎么办？
如果样本 A₁<1.5 或ΔA>0.7，样本需用 PBS 适当进行稀释后，再进行实验，或减少提取用样本量。如果样本的 A₁>2.0 且ΔA<0.03，可适当提高样本量或延长检测时间。测定肝脏样本时建议稀释倍数为 20-80 倍，测定肾脏样本时建议稀释倍数为 20-40 倍，测定心脏样本时建议稀释倍数为 40-100 倍。
Aspartate aminotransferase (AST, formerly known as glutamic-oxaloacetic transaminase, GOT, EC 2.6.1.1) is widely distributed in animal and plant tissues and is mainly present in the liver, myocardium, skeletal muscle, and kidneys. When tissue cells are damaged and ruptured, large amounts of the enzyme are released into body fluids. Therefore, AST activity is an important biochemical indicator for evaluating the degree of tissue cell damage.
The detection principle of this kit is that AST in the sample catalyzes the transamination reaction between L-aspartate and α-ketoglutarate to generate oxaloacetate and L-glutamate. Malate dehydrogenase (MDH) reduces the generated oxaloacetate to L-malate while consuming NADH and generating NAD⁺. NADH exhibits strong characteristic ultraviolet absorbance at 340 nm, whereas NAD⁺ shows almost no absorbance at this wavelength. Therefore, AST activity can be calculated by measuring the rate of decrease in absorbance at 340 nm.
Contents & Storage:
* Please check the volume of each component before use. * Each component is supplied with an additional 10% volume beyond the specified amount for standard curve preparation or preliminary experiments. |
Self-supplied Instruments and Reagents:
| Type of Self-supplied Resources | Resource Name | Remarks |
|---|---|---|
| Instruments | Microplate reader | Capable of measuring absorbance at 340 nm |
| Consumables | 96-well microplate | UV plate |
| Reagents | PBS | For sample extraction/dilution. |
| Others | Homogenizer (for tissue), ultrasonic disruptor (for cells or bacteria), incubator, low-temperature centrifuge, adjustable-volume pipettes and pipette tips. | A multi-channel pipette is recommended to boost experimental efficiency when processing a large number of test samples. |
Precautions:
1. Before formal testing, it is recommended to select 2-3 samples expected to show relatively large differences for a preliminary experiment.
2. For tissue samples, cell samples, etc., results among samples can be normalized by measuring the protein concentration. The Aladdin BCA Protein Assay Kit, Catalog No. B665595, is recommended.
3. This kit is compatible with spectrophotometric detection. Please adjust the preparation volume of the detection reagents proportionally according to the requirements for spectrophotometric detection.
4. Biochemical assay reagents are generally irritating and biologically toxic. For your safety and health, please take appropriate biosafety precautions throughout the experiment, wear protective equipment such as a lab coat, mask, gloves, and head covering, and perform the experiment in a fume hood or biosafety cabinet.
5. This product is for scientific research use only and is not intended for clinical diagnosis.
Experimental Procedures:
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Precautions |
|---|---|---|
| Reagent Ⅰ | Ready-to-use; equilibrate to room temperature before use | Store at 2-8℃ |
| Reagent Ⅱ | Ready-to-use; centrifuge at low speed before use and equilibrate to room temperature | Store at 2-8℃ |
| Reagent Ⅲ | Ready-to-use; equilibrate to room temperature before use | Store at 2-8℃ |
2. Sample Preparation
Note: Fresh samples are recommended. Samples should be extracted as freshly as possible. If extraction cannot be performed on the same day, intact cells or aliquoted tissue blocks should be stored at -80°C and used within one month. When ready to perform the experiment, remove the samples from -80°C and thaw them on ice. However, please note that this may affect sample stability, and the experimental results may be lower than expected.
2.1 Animal and plant tissues: Weigh approximately 0.1 g of tissue sample, add 1 mL PBS, homogenize in an ice bath, centrifuge at 12000 g and 4°C for 10 min, collect the supernatant, and keep it on ice until testing.
2.2 Cells or bacteria: Collect 5×10⁶ cells or bacteria, wash the cells or bacteria with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Add 1 mL PBS and ultrasonically disrupt the cells or bacteria in an ice bath for 5 min (power: 20% or 200 W; sonication for 3 s, interval for 7 s, repeated 30 times), centrifuge at 12000 g and 4°C for 10 min, collect the supernatant, and keep it on ice until testing.
2.3 Liquid samples such as serum (plasma): Test directly.
3. Experimental Procedures
3.1 Microplate reader preparation: Preheat for more than 30 min and set the wavelength to 340 nm.
3.2 Preparation of Working Solution: Prepare the Working Solution at 200 μL per well (Reagent Ⅰ 200 μL : Reagent Ⅱ 0.2 μL, volume ratio 1000:1), mix thoroughly, and prepare fresh before use.
3.3 Preparation of the assay system: Operate in a 96-well plate as follows:
| Reagent | Assay Well (μL) |
|---|---|
| Sample | 20 |
| Working Solution | 200 |
| Mix well and incubate at 37°C for 5 min | |
| Reagent Ⅲ | 80 |
3.4 Absorbance measurement: Mix well and record the absorbance value A₁ at 340 nm after incubation at 37°C for 5 min and the absorbance value A₂ at 10 min.
Calculation of Results
The derivation formula and the simplified calculation formula provided below are mathematically equivalent.
1. Data Processing
Calculate ΔA=A₁-A₂.
2. Calculation of AST Activity
2.1 Calculation Based on Protein Concentration:
Enzyme activity definition: At 37°C, the amount of enzyme that consumes 1 μmol NADH per minute in the reaction system per g of tissue protein is defined as one unit of enzyme activity.
AST (U/g prot)=[ΔA÷(ε×d)×10⁶×Vtotal reaction]÷(Vsample×Cpr)÷T=535.9×ΔA÷Cpr
2.2 Calculation Based on Sample Fresh Weight:
Enzyme activity definition: At 37°C, the amount of enzyme that consumes 1 μmol NADH per minute in the reaction system per g of tissue is defined as one unit of enzyme activity.
AST (U/g)=[ΔA÷(ε×d)×10⁶×Vtotal reaction]÷(W×Vsample÷Vtotal sample)÷T=0.5359×ΔA÷W
2.3 Calculation Based on the Number of Cells or Bacteria:
Enzyme activity definition: At 37°C, the amount of enzyme that consumes 1 μmol NADH per minute in the reaction system per 10⁶ cells or bacteria is defined as one unit of enzyme activity.
AST (U/10⁶)=[ΔA÷(ε×d)×10⁶×Vtotal reaction]÷(N×Vsample÷Vtotal sample)÷T=0.5359×ΔA÷N
2.4 Calculation Based on Liquid Volume:
Enzyme activity definition: At 37°C, the amount of enzyme that consumes 1 μmol NADH per minute in the reaction system per L of liquid sample is defined as one unit of enzyme activity.
AST (U/L)=[ΔA÷(ε×d)×10⁶×Vtotal reaction]÷Vsample÷T=535.9×ΔA ε: Molar extinction coefficient of NADH, 6.22×10³ L/mol/cm; d: optical path length of the 96-well UV plate, 0.9 cm; 10⁶: unit conversion factor, 1 mol=10⁶ μmol; Vtotal reaction: total volume of the reaction system, 3×10⁻⁴ L; Vsample: volume of sample added to the reaction system, 2×10⁻⁵ L; Cpr: sample protein concentration, g/L; T: reaction time, 5 min; Vtotal sample: volume of PBS added, 10⁻³ L; W: sample mass, g; N: number of cells, in units of 10⁶ (for example, if the number of cells is 5×10⁶, N=5).
Results Presentation:
Example 1: Take 0.1 g of mouse heart tissue and perform the procedure as described above. After diluting the sample 90-fold with PBS, measure it using a 96-well UV plate to obtain ΔA=A₁-A₂=2.190-2.007=0.183. Based on the sample fresh weight, the AST activity of the 90-fold diluted mouse heart sample is calculated as follows: AST (U/g)=0.5359×ΔA÷W=0.981 U/g.
Frequently Asked Questions:
What should I do if the measured sample ΔA is too high or too low?
If the sample A₁<1.5 or ΔA>0.7, dilute the sample appropriately with PBS before repeating the experiment, or reduce the amount of sample used for extraction. If the sample A₁>2.0 and ΔA<0.03, the sample amount may be appropriately increased or the detection time may be extended. For liver samples, a dilution factor of 20-80 is recommended; for kidney samples, a dilution factor of 20-40 is recommended; and for heart samples, a dilution factor of 40-100 is recommended.
* 请在实验前检查各组分的量。 * 各组分在规格基础上额外提供 10% 的量用于进行标准曲线制作或预实验。 |
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