天冬氨酸转氨酶 (AST），亦称为血清谷氨酸草酰乙酸转氨酶 (GOT) 或天冬氨酸氨基转移酶 (ASAT/AAT），可催化天冬氨酸与 α‑酮戊二酸转化生成草酰乙酸和谷氨酸。人体存在两种同工酶：GOT1 为胞质同工酶，来源于红细胞与心脏；GOT2 为线粒体同工酶，主要存在于肝脏。肝脏及肌肉相关疾病会引起 AST 水平升高。AST 是肝功能、心肌梗死、急性胰腺炎、急性溶血性贫血、重度烧伤、急性肾病及创伤等临床诊断检测的指标之一。简便、直接且可适配自动化设备的 AST 活性检测方案，在科研与药物研发领域应用广泛。

本天冬氨酸转氨酶 (AST) 检测试剂盒基于对 AST 催化生成的草酰乙酸进行定量检测。检测体系中，苹果酸脱氢酶催化草酰乙酸与 β‑NADH 转化为苹果酸与 NAD⁺。340 nm 处 NADH 吸光度的下降值与 AST 活性成正比。

本试剂盒可采用紫外检测法，具有检测灵敏度高、线性范围宽、样品用量少等特点。在样品体积为20 µL时，可检测范围为 2‑100 U/L酶活样本。适用于血清、血浆及其他生物样本的 AST 活性测定，也可用于研究药物对 AST 活性的影响。

组分货号	组分名称	物理外观	100 T	储存	单次测试使用量
A1522585A	检测缓冲液	液体	25.0 mL	-20℃.避光	196.0 μL
A1522585B	辅因子	液体	240.0 μL	-20℃.避光	2.0 μL
A1522585C	酶液	液体	240.0 μL	-20℃.避光	2.0 μL
A1522585D	NADH 试剂	粉末	7.17 mg	-20℃.避光	按需

使用方法

1. 试剂盒的准备

试剂开盖前简短离心；所有组分使用前平衡至检测温度 (室温或 37 ℃）。

1) NADH 试剂 :向瓶内加入 1000 µL 纯化水复溶，配制成 10 mM NADH工作液。复溶后的 10 mM NADH工作液 ‑20 ℃冷冻保存可稳定3周左右。

2) 检测缓冲液：即用型；使用前充分剧烈振荡混匀。

3) 酶混合液：即用型；融化后置于冰上保存。

4) 辅因子：即用型；融化后置于冰上保存。

2. 样本制备

推荐使用新鲜样本，若不立即实验，-80℃可保存1个月，使用时冰上解冻。

1) 动物组织用冷 PBS 清洗组织，尽可能去除血液。吸干水分，称取 0.1 g，加入 1 mL 预冷的裂解液，冰浴匀浆。10000 g，4℃离心 5 min，取上清置冰上待测。

2) 植物组织称取约 0.1 g 样本，加入 1 mL 预冷的裂解液捣碎，冰浴超声破碎 5 min (功率 20% 或 200 W，超声 3 s，间隔 7 s，重复 30 次），10000 g，4℃离心 5 min，取上清置冰上待测。

3) 细胞 (菌) 收集每 1×10⁶ 个细胞或细菌，用冷 PBS 清洗后，加入 0.1-0.2 mL 预冷的裂解液冰浴匀浆或超声破碎 5 min。12000 g，4℃离心 5 min，取上清置冰上待测。

4) 血浆、血清、尿液等生物体液去除蛋白后取上清：可使用 10 kDa 超滤管过滤取滤液；或将样本：30% ZnSO₄溶液 = 20:1 混合涡旋，10000 g 室温离心 5 min，取上清，(溶血样本不可使用)。

注：裂解液需自行制备，不影响酶活检测即可，可使用PBS。

3. 反应工作液的配制

1) 根据检测总孔数配制足量试剂，按照下表配制工作试剂。

试剂 (µL)	检测工作试剂	空白工作试剂
检测缓冲液	196	196
辅因子	2	2
酶液	2	2
10 mM NADH工作液	6	-
纯化水	-	6

2) 将配制好的工作试剂预热至目标实验温度。

3) 取待测样品各20 µL,向样本孔、NADH 标准孔各加入 200 µL 检测工作试剂；向空白孔加入 200 µL 空白工作试剂；轻敲微孔板混匀,参照下表进行添加：

加入物 (µL)	样品孔	空白孔	NADH标准孔
检测工作试剂	200	-	200
空白工作试剂	-	200	-
纯化水	-	20	20
待测样本	20	-	-

4. 样本中酶活性测定

1) 酶标仪开机预热 30 min 以上，调节波长 340 nm, 将微孔板置于目标实验温度（如 37 ℃）。

1) 立即轻敲板子混匀；在设定温度下孵育，分别读取 5 min、10 min 时 340 nm 处光密度值（OD）；也可使用动力学模式持续记录 340 nm 光密度。

5. 结果计算

1) 对每个样本，用 5 min 光密度减去 10 min 光密度，计算 NADH 消耗速率，记为 ΔODₛ。

2) 对 NADH 标准孔，用 5 min 光密度减去 10 min 光密度，得到标准品的变化速率 ΔODₛₜₐₙdₐᵣd 。

3) 96 孔板实验，采用下面公式计算 AST 活性：

AST(U/L)=583 × (ΔODₛ − ΔODₛₜₐₙdₐᵣd ) / (ODₛₜₐₙdₐᵣd− ODbₗₐₙₖ ))

系数推导：

ODₛₜₐₙdₐᵣd = 5 minute OD reading at 340 nm of the NADH Standard

ODbₗₐₙₖ= 5 minute OD reading at 340 nm of the Blank

583 (系数)=10 mM NADH×(6 μL (NADH 体积) / 206 μL (工作试剂体积)) × (200 μL (工作试剂加入体积) / 220 μL (体系总容积)) × (11 (样本稀释倍数) / 5 分钟) = 583 μM/min

注：若计算得到的 AST 活性＞100 U/L，需用检测缓冲液稀释样本后重新测定，最终结果乘以稀释倍数。

酶活单位定义:1 单位 (U) AST，pH 8.0条件下，每分钟催化 1 微摩尔天冬氨酸转化为草酰乙酸。

注意事项

1) 试剂光敏、易氧化，全程避光，开瓶配制后尽快使用。

2) 试剂盒干冰 / 湿冰运输；所有组分长期储存条件：‑20 ℃保存。

3) 对于酶活较低的样品可延长检测时间，选择合适的时间窗口进行酶活计算。

4) 为了您的安全和健康，请穿实验服并戴一次性手套操作。

5) 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品。

附录：

AST 酶的反应示例（37 ℃检测）

注：图纵坐标OD₃₄₀，横坐标时间min；NADH Standard标准组OD几乎不变；AST (80 U/L) 组OD随时间下降；Blank空白组OD维持低位。

Aspartate Transaminase (AST), also known as serum glutamic oxaloacetic transaminase (GOT) or aspartate aminotransferase (ASAT/AAT), facilitates the conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate. There are two isoenzymes in humans: GOT1 is a cytosolic isoenzyme derived from red blood cells and heart; GOT2 is the mitochondrial isoenzyme found mainly in the liver. AST is elevated in liver and muscle diseases. It is part of diagnostic testing for liver function, myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, and trauma. Simple, direct and automation-ready procedures for measuring AST activity find wide applications in research and drug discovery. The Aspartate Transaminase (AST) Assay Kit is based on the quantification of oxaloacetate produced by AST. In this assay, oxaloacetate and NADH are converted to malate and NAD+ by the enzyme malate dehydrogenase. The decrease in NADH absorbance at 340 nm is proportionate to AST activity.

This kit adopts a UV‑detection method and features high detection sensitivity, broad linear range and low sample consumption. With a sample volume of 20 µL, it can detect samples with enzyme activities ranging from 2‑100 U/L. It is suitable for the determination of AST activity in serum, plasma and other biological samples, and can also be used to investigate the effects of drugs on AST activity.

Component No.	Component Name	Appearance	100 T	Storage	Quantity Per Test
A1522585A	Assay Buffer	Liquid	25.0 mL	-20℃.Store in the dark.	196.0 μL
A1522585B	Cofactor	Liquid	240.0 μL	-20℃.Store in the dark.	2.0 μL
A1522585C	Enzyme	Liquid	240.0 μL	-20℃.Store in the dark.	2.0 μL
A1522585D	NADH	Power	7.17 mg	-20℃.Store in the dark.	As needed

Usage Method

1. Kit Preparation

Briefly centrifuge small‑volume reagent vials before opening. Equilibrate all kit components to assay temperature (room temperature or 37 °C) prior to use.

1) NADH: Reconstitute the vial with 1000 µL of purified water to prepare a 10 mM NADH working reagent . The reconstituted 10 mM NADH working reagent is stable for approximately 3 weeks when stored frozen at -20 °C.

2) Assay Buffer: Ready‑to‑use. Vortex thoroughly before use.

3) Enzyme Mix: Ready‑to‑use. Keep thawed reagent on ice.

4) Cofactor: Ready‑to‑use. Keep thawed reagent on ice.

2. Sample Preparation

Fresh samples are recommended. If assays cannot be performed immediately, samples may be stored at -80 °C for up to 1 month. Thaw samples on‑ice before use.

1) Animal tissue: Rinse tissue with cold PBS to remove residual blood as much as possible. Blot dry, weigh 0.1 g tissue, add 1 mL pre‑chilled lysis buffer, and homogenize on ice. Centrifuge the homogenate at 10 000 × g for 5 min at 4 °C. Collect the supernatant and keep on‑ice for assay.

2) Plant tissue: Weigh approximately 0.1 g sample, add 1 mL pre‑chilled lysis buffer and crush the tissue. Perform sonication on ice for 5 min (power output: 20 % or 200 W; 3 s sonication / 7 s interval, 30 cycles total). Centrifuge at 10 000 × g for 5 min at 4 °C. Collect the supernatant and keep on‑ice for assay.

3) Cells / Bacteria: Collect 1 × 10⁶ cells or bacteria. Wash with cold PBS, add 0.1‑0.2 mL pre‑chilled lysis buffer, then homogenize or sonicate on ice for 5 min. Centrifuge at 12 000 × g for 5 min at 4 °C. Collect the supernatant and keep on‑ice for assay.

4) Biological fluids (plasma, serum, urine): Deproteinize samples and collect supernatant. Two options are available: filter with 10 kDa MWCO ultrafiltration spin columns and collect the filtrate; or mix sample with 30 % ZnSO₄ solution at a volume ratio of 20:1, vortex thoroughly, centrifuge at 10000× g for 5 min at room temperature, and collect the supernatant.(Note: Hemolyzed samples are not suitable for testing).

Note: Lysis buffer must be prepared independently. Any buffer compatible with enzyme‑activity measurement may be used; PBS is an acceptable option.

3. Preparation of Working Reaction Reagent

1) Prepare sufficient reagents according to the total number of assay wells. Prepare working reagents following the table below.

Reagent (µL)	Assay Working Reagent	Blank Working Reagent
Assay Buffer	196	196
Cofactor	2	2
Enzyme	2	2
10 mM NADH working Reagent	6	-
Purified Water	-	6

2) Warm the prepared working reagent to the target assay temperature.

3) Transfer 20 µL of each test sample. Add 200 µL of Assay Working Reagent to sample wells and NADH standard wells; add 200 µL of Blank Working Reagent to blank wells. Tap the microplate gently to mix. Add reagents according to the table below:

Reagent Volume (µL)	Sample well	Blank well	NADH standard well
Assay Working Reagent	200	-	200
Blank Working Reagent	-	200	-
Purified Water	-	20	20
Sample	20	-	-

4. Measurement of AST Activity in Samples

1) Power on the microplate reader and preheat for no less than 30 min. Set the wavelength to 340 nm, and maintain the microplate at the target assay temperature ( 37 °C).

2) Tap the plate immediately to mix thoroughly. Incubate at the set temperature, and record optical density (OD) at 340 nm at 5 min and 10 min respectively. Alternatively, use kinetic mode for continuous OD recording at 340 nm.

5. Calculation of Results

1) For each sample, subtract the OD value at 10 min from the OD value at 5 min to calculate the NADH consumption rate, denoted as ΔODₛ .

2) For the NADH standard well, subtract the OD value at 10 min from the OD value at 5 min to obtain the change rate of the standard, denoted as ΔOD<sub> standard </sub>.

3) For 96‑well plate assays, calculate AST activity using the formula below:

Derivation of the factor:

AST(U/L)=583 × (ΔODₛ−ΔODₛₜₐₙdₐᵣd ) / (ODₛₜₐₙdₐᵣd−ODbₗₐₙₖ ))

ODₛₜₐₙdₐᵣd = 5 minute OD reading at 340 nm of the NADH Standard

ODbₗₐₙₖ= 5 minute OD reading at 340 nm of the Blank

Factor 583=10 mM NADH ×(6 μL (NADH volume) / 206 μL ( working reagent volume )) × (200 μL ( added working reagent volume ) / 220 μL (total reaction volume )) × (11 (sample dilution factor ) / 5 min) = 583 μM/min

If the calculated AST activity is >100 U/L, dilute the sample with Assay Buffer and re‑measure. Multiply the final result by the dilution factor.

Definition of enzyme activity unit: One unit (U) of AST is defined as the amount of enzyme that catalyzes the conversion of 1 µmol aspartate to oxaloacetate per minute at pH 8.0.

Precautions

1) The reagents are light‑sensitive and susceptible to oxidation. Keep away from light throughout the experiment and use promptly after vial opening and reconstitution.

2) The kit is shipped on dry ice / wet ice. For long‑term storage, keep all components at -20 °C.

3) For samples with low enzyme activity, extend the assay duration and select an appropriate time window for enzyme‑activity calculation.

4) For your safety and health, wear a lab coat and disposable gloves during operation.

5) This product is for scientific research use only by qualified professionals. It shall not be used for clinical diagnosis or therapy, nor for food or pharmaceutical applications.

Appendix:

Example of AST enzymatic reaction (assayed at 37 ℃)

Note: Y‑axis of the figure: OD₃₄₀ ; X‑axis: time (min). The OD value of the NADH Standard group remains nearly constant; the OD value of the AST (80 U/L) group decreases over time; the OD value of the Blank group stays at a low level.

规格

别名

天门冬氨酸氨基转移酶检测试剂盒 (天门冬氨酸底物法），天门冬氨酸氨基转移酶测试盒

英文别名

Aspartate Transaminase (AST) Assay Kit

规格或纯度

BioReagent, 比色法, 适用于分析

英文名称

Aspartate Transaminase (AST/GOT) Assay Kit (UV Micro Method)

应用

细胞代谢

检测范围

0-100 U/L

检测方法

紫外微量法

检测仪器

酶标仪

检测波长

40 nm

检测时长

30 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

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C of A & Other Certificates(BSE/TSE, COO):

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技术文档和文章

a1522585 天门冬氨酸氨基转移酶 astgot 活性检测测试盒紫外微量法.pdf

a1522585 aspartate transaminase astgot assay kit uv micro method.pdf

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

浓度

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分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

体积 1 (V1)

浓度 2 (C2)

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

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天门冬氨酸氨基转移酶 (AST/GOT) 活性检测测试盒 (紫外微量法）