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。
动物肝脏、肾脏是氨基酸代谢的主要器官，故尿中氨基酸的变化最能反应肝、肾的生理状态。另外，氨基酸还能反应灼伤、伤寒等方面情况。植物体内氨基酸含量对研究植物在不同条件下及不同生长发育时期氮代谢变化、植物对氮素的吸收、运输、同化及营养状况等有重要意义。
检测原理：氨基酸的α-氨基可与水合茚三酮反应，产生蓝紫色化合物，在570nm处有特征吸收峰；通过测定570 nm吸光度来计算氨基酸含量。
适用样本：血清（浆）、动植物组织、细胞、细胞上清、细菌、尿液。
注：
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。
自备仪器和试剂
操作步骤（仅供参考）
1. 试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| 提取液 | 即用型；使用前平衡到室温 | 4℃保存 |
| 检测缓冲液 | 即用型；使用前平衡到室温 | 4℃保存 |
| 工作底物辅助因子 | 临用前配制；取一瓶Substrate Cofactor加入2.5 mL 检测缓冲液充分溶解 | 现配现用；有毒且有刺激性，建议在使 用过程中做好个人防护 |
| 工作底物 | 临用前配制；48T加入2 mL 95%乙醇充分溶解， 96T加入4 mL 95%乙醇充分溶解 | 未用完的试剂可4℃避光保存1周；有毒且有刺激性，建议在使用过程中做好个人防护 |
| 100 µmol/mL 标准品 | 临用前配制，加1.332 mL去离子水，充分溶解得到100 μmol/mL甘氨酸标准品 | 未用完的试剂可4℃避光保存1个月 |
2. 标准品的配制
使用100 µmol/mL 标准品，按照下表所示，进一步稀释标准品。每次实验都要做一次标准品检测，制作标曲；稀释后的标准品溶液不稳定，必须在4小时内使用。
| 序号 | 标准品（μL） | 提取液（μL） | 浓度（μmol/mL） |
| 1 | 25 µL of 100 μmol/mL | 975 | 2.5 |
| 2 | 100 µL of 2.5 μmol/mL | 100 | 1.25 |
| 3 | 100 µL of 1.25 μmol/mL | 100 | 0.625 |
| 4 | 100 µL of 0.625 μmol/mL | 100 | 0.3125 |
| 5 | 100 µL of 0.3125 μmol/mL | 100 | 0.15625 |
| 6 | 100 µL of 0.15625 μmol/mL | 100 | 0.078125 |
| 空白 | 0 | 200 | 0 |
3. 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。
3.1 动物组织
称取约0.1 g动物组织样本，加入1 mL 提取液，室温充分匀浆，转移到1.5 mL EP管中（推荐使用螺旋盖EP管），盖紧后（防止水分散失）置于沸水浴15 min；自来水冷却后，10000 g，室温离心10 min，取上清液，待测。
3.2 植物组织
称取约0.1 g植物组织样本，加入1 mL 提取液捣碎，室温超声波破碎5 min（功率20%或200W，超声3 s，间隔7 s，重复30次），转移到1.5 mL EP管中（推荐使用螺旋盖EP管）， 盖紧后（防止水分散失）置于沸水浴15 min；自来水冷却后，10000 g，室温离心10 min，取上清液， 待测。
3.3 细菌或细胞
收集5×10⁶个细菌或细胞，用冷PBS清洗细菌或细胞，800 g离心2 min，弃上清液。加入1 mL 提取液，室温超声波破碎细菌或细胞5 min（功率20%或200 W，超声3 s，间隔7 s，重复30次），转移到1.5 mL EP管中（推荐使用螺旋盖EP管），盖紧后（防止水分散失）置于沸水浴15 min；自来水冷却后，10000 g，室温离心10 min，取上清液，待测。
3.4 血清（浆）、细胞上清、尿液等液体
吸取0.5 mL液体加入0.5 mL 提取液（推荐使用螺旋盖EP管），盖紧后（防止水分散失）置于沸水浴15 min；自来水冷却后，10000 g，室温离心10 min，取上清液，待测。
4. 实验步骤
4.1 酶标仪准备
预热30 min以上，调节波长到570 nm。
4.2 检测体系配制
在1.5 mL EP管（推荐使用螺旋盖EP管）中按照如下方式操作。
| 试剂 | 空白管（μL） | 标准管（μL） | 测定管（μL） |
| 提取液 | 10 | 0 | 0 |
| 标准品 | 0 | 10 | 0 |
| 样本 | 0 | 0 | 10 |
| 工作底物辅助因子 | 50 | 50 | 50 |
| 工作底物 | 20 | 20 | 20 |
4.3 混匀后盖紧管盖（防止水分散失），置于沸水浴中保温5 min，冰浴冷却30 s后加入120 μL 60%乙醇，反复颠倒EP管数次，每管取出150 μL加入96孔板中。
4.4 吸光度检测
于570 nm处测定吸光值，分别记为A空白、A标准和A测定，显色后务必在30 min内测完。
5. 结果计算
5.1 数据处理
计算ΔA测定=A测定-A空白，ΔA标准=A标准-A空白。
5.2 标准曲线绘制
以标准品浓度为y轴，ΔA标准为x轴，绘制标准曲线。将ΔA测定代入方程得到y值（μmol/mL）。
5.3 样本氨基酸含量的计算
(1) 按样本质量计算 氨基酸含量 (μmol/g)=y÷(W÷V提取)×n=y÷W×n
(2) 按细菌或细胞数量计算 氨基酸含量 (μmol/10⁴)=y÷(N÷V提取)×n=y÷N×n
(3) 按液体体积计算 氨基酸含量 (μmol/mL)=y×2×n
(4) 按蛋白浓度计算 氨基酸含量 (μmol/mg prot)=y÷Cpr×n
参数说明：
W：样本质量，g；
V提取：加入 提取液体积，1 mL；
n：样本的稀释倍数；
Cpr：上清液蛋白质浓度，mg/mL；
N：细菌或细胞数量，以10⁴为单位（例如细菌数量为5×10⁶，N=500）；
2：提取液体时的稀释倍数，(0.5 mL+0.5 mL)/0.5 mL=2。
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B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3. 脯氨酸和羟脯氨酸与茚三酮反应在570 nm处无吸收峰，因此，570 nm处测定结果不含这两种氨基酸的量。
4. 建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。
5. 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。
6. 本产品仅供科学研究使用，不适用于临床诊断。
结果展示
典型标准曲线：y=2.8806x-0.0054，R²=0.9998
示例：取0.1 g绿萝叶，按照测定步骤操作，用96孔板进行检测。测得ΔA测定=A测定-A空白=0.295-0.065=0.23，代入标准曲线，计算y=0.657。按样本质量计算得：氨基酸含量(μmol/g)=y÷(W÷V提取)×n=y÷W×n=6.57μmol/g。
常见问题及解答
Q：检测得到的样本A测定过高或者过低怎么办？
A：如果样本A测定>2.5 μmol/mL的ΔA标准，样本可用去离子水进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。如果样本的A测定<0.01，可适当提高样本量。
Animal liver and kidneys are the primary organs for amino acid metabolism; therefore, changes in urinary amino acids most accurately reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. In plants, the amino acid content is significant for studying nitrogen metabolism changes under different conditions and at various growth stages, as well as for understanding nitrogen absorption, transport, assimilation, and nutritional status.
Detection Principle
The α-amino group of amino acids reacts with ninhydrin to produce a blue-purple compound with a characteristic absorption peak at 570 nm. The amino acid content is calculated by measuring the absorbance at 570 nm.
Applicable Samples: Serum (plasma), animal and plant tissues, cells, cell supernatants, bacteria, urine.
Note:
Please check the volume of each component before starting the experiment.
Each component provides an additional 10% beyond the specified amount for standard curve preparation or pilot experiments.
Reagents, consumables and Equipments not provided
Assay Procedure (For Reference Only)
1. Reagent Preparation
| Reagent Name | Preparation Procedure | 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. |
| Working Substrate Cofactor | Prepare immediately before use. Add 2.5 mL of Assay Buffer to one vial of Substrate Cofactor and dissolve completely. | Use immediately. Toxic and irritant; recommended to wear personal protective equipment during use. |
| Working Substrate | Prepare immediately before use. For 48T, add 2 mL of 95% ethanol to dissolve completely. For 96T, add 4 mL of 95% ethanol to dissolve completely. | Unused portion can be stored at 4°C protected from light for up to 1 week. Toxic and irritant; recommended to wear personal protective equipment during use. |
| 100 µmol/mL Standard | Prepare immediately before use. Add 1.332 mL of deionized water to dissolve completely to obtain a 100 µmol/mL Glycine Standard. | Unused portion can be stored at 4°C protected from light for up to 1 month. |
2. Preparation of Standards
Use the 100 µmol/mL Standard and dilute further according to the table below. Prepare a standard curve for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
|
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.
3.1 Animal Tissues
Weigh approximately 0.1 g of animal tissue. Add 1 mL of Extraction Buffer and homogenize thoroughly at room temperature.
Transfer to a 1.5 mL screw-cap microcentrifuge tube. Cap tightly (to prevent evaporation) and place in a boiling water bath for 15 min.
Cool under running tap water. Centrifuge at 10,000 g for 10 min at room temperature. Collect the supernatant for analysis.
3.2 Plant Tissues
Weigh approximately 0.1 g of plant tissue. Add 1 mL of Extraction Buffer and macerate.
Sonicate at room temperature for 5 min (power 20% or 200W, 3s pulse on, 7s pulse off, 30 cycles). Transfer to a 1.5 mL screw-cap microcentrifuge tube.
Cap tightly and place in a boiling water bath for 15 min.
Cool under running tap water. Centrifuge at 10,000 g for 10 min at room temperature. Collect the supernatant for analysis.
3.3 Bacteria or Cells
Collect 5×10⁶ bacteria or cells. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant.
Add 1 mL of Extraction Buffer. Sonicate at room temperature for 5 min (power 20% or 200W, 3s pulse on, 7s pulse off, 30 cycles). Transfer to a 1.5 mL screw-cap microcentrifuge tube.
Cap tightly and place in a boiling water bath for 15 min.
Cool under running tap water. Centrifuge at 10,000 g for 10 min at room temperature. Collect the supernatant for analysis.
3.4 Liquid Samples (Serum/Plasma, Cell Supernatant, Urine)
Pipette 0.5 mL of the liquid sample into a 1.5 mL screw-cap microcentrifuge tube. Add 0.5 mL of Extraction Buffer.
Cap tightly and place in a boiling water bath for 15 min.
Cool under running tap water. Centrifuge at 10,000 g for 10 min at room temperature. Collect the supernatant for analysis.
4. Assay Procedure
4.1 Microplate Reader Preparation
Pre-heat the microplate reader for at least 30 min and set the wavelength to 570 nm.
4.2 Reaction Setup
Prepare the reactions in 1.5 mL screw-cap microcentrifuge tubes as follows:
|
4.3 Incubation and Transfer
Mix well. Cap the tubes tightly (to prevent evaporation). Place in a boiling water bath for 5 min.
Cool in an ice bath for 30 s. Add 120 µL of 60% ethanol. Invert the tubes several times to mix.
Transfer 150 µL from each tube into a well of a 96-well microplate.
4.4 Absorbance Measurement
Measure the absorbance at 570 nm. Record the values as ABlank, AStandard, and ATest. The measurement must be completed within 30 min after color development.
5. Result Calculation
5.1 Data Processing
Calculate ΔATest = ATest - ABlank
Calculate ΔAStandard = AStandard - ABlank
5.2 Standard Curve
Plot the standard concentration (y-axis, µmol/mL) against the corresponding ΔAStandard (x-axis) to generate a standard curve. Obtain the linear equation y = ax + b. Substitute the ΔATest value into the equation to obtain the concentration y (µmol/mL) for the sample.
5.3 Calculation of Sample Amino Acid Content
(1) By Sample Mass (Tissue):
Amino Acid Content (µmol/g) = y ÷ (W ÷ Vextraction) × n = y ÷ W × n
(2) By Cell/Bacteria Count:
Amino Acid Content (µmol/10⁴ cells) = y ÷ (N ÷ Vextraction) × n = y ÷ N × n
(3) By Liquid Volume (Serum, Urine, etc.):
Amino Acid Content (µmol/mL) = y × 2 × n
(4) By Protein Concentration:
Amino Acid Content (µmol/mg prot) = y ÷ Cpr × n
Parameter Explanation:
y: Amino acid concentration from standard curve (µmol/mL).
W: Sample mass (g).
Vextraction: Volume of Extraction Buffer used for extraction (here, 1 mL).
n: Dilution factor of the sample before addition to the reaction.
N: Number of bacteria or cells, in units of 10⁴ (e.g., for 5×10⁶ cells, N = 500).
2: Dilution factor for liquid samples: (0.5 mL sample + 0.5 mL buffer) / 0.5 mL sample = 2.
Cpr: Protein concentration of the sample supernatant (mg/mL).
Notes
Pilot Experiment: Before the formal assay, it is recommended to perform a pilot experiment using 2-3 samples with expected significant differences.
Normalization: For tissue and cell samples, results can be normalized by measuring the protein concentration. BCA Protein Assay Kits are recommended.
Assay Specificity: Proline and hydroxyproline do not produce an absorption peak at 570 nm when reacting with ninhydrin. Therefore, the results measured at 570 nm do not include these two amino acids.
Standard Curve: It is recommended to establish your own standard curve for the most accurate results. If not, the typical standard curve equation provided in the results section can be used as a reference.
Safety: Biochemical detection reagents are generally irritants and have biological toxicity. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, head cover) and perform experiments in a fume hood or biosafety cabinet throughout the process.
Intended Use: This product is for scientific research only and is not intended for clinical diagnosis.
Results Display
Typical Standard Curve: y = 2.8806x - 0.0054, R² = 0.9998
Example:
0.1 g of Epipremnum aureum leaf was processed according to the procedure and measured in a 96-well plate.
ΔATest = ATest - ABlank = 0.295 - 0.065 = 0.23.
Substituting into the standard curve: y = (2.8806 * 0.23) - 0.0054 = 0.657.
Calculated by sample mass: Amino Acid Content (µmol/g) = y ÷ (W ÷ Vextraction) × n = y ÷ W × n = 0.657 ÷ 0.1 × 1 = 6.57 µmol/g.
Frequently Asked Questions (FAQ)
Q: What should I do if the measured ATest is too high or too low?
A:
If ATest is higher than the ΔAStandard of the 2.5 µmol/mL standard (> approx. 2.5 µmol/mL equivalent), dilute the sample further with deionized water, multiply the result by the dilution factor, or reduce the amount of sample used for extraction.
If ATest is < 0.01, consider increasing the sample volume appropriately.
| Product number | Component | 48T | 96T | Storage |
| A1519780A | 提取液 | 70 mL | 70 mL×2 | 2-8℃. |
| A1519780B | 检测缓冲液 | 7.5 mL | 15 mL | 2-8℃. |
| A1519780C | 底物辅助因子 | 2 EA | 4 EA | 2-8℃. |
| A1519780D | 底物 | 1 EA | 1 EA | 2-8℃. Store in the dark. |
| A1519780E | 标准品 | 1 EA | 1 EA | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737038 |
分析证书 | A1519780 |
| ZJ26F0737037 |
分析证书 | A1519780 |
| ZJ26F0535159 |
分析证书 | A1519780 |
| ZJ26F0434539 |
分析证书 | A1519780 |
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