BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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。
赖氨酸是人体必需氨基酸之一，能促进人体发育、增强免疫功能，并有提高中枢神经组织功能的作用。赖氨酸为碱性必需氨基酸。由于谷物食品中的赖氨酸含量甚低，且在加工过程中易被破坏而缺乏，故称为第一限制性氨基酸。
检测原理：蛋白质中的赖氨酸具有一个游离的ε-NH₂，它与茚三酮试剂反应生成蓝紫色物质，在570nm有特征吸收峰；通过测定570nm吸光度，来计算赖氨酸含量。
检测范围：0.125-8 μmol/mL
灵敏度：0.03125 μmol/mL
适用样本：血清；血浆；动植物组织；细胞；细胞上清；细菌
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
操作步骤
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 |
| Working Substrate | 临用前配制，96T加入2mL 95%乙醇充分溶解 | 未用完的Working Substrate可4℃避光保存1周 |
| Working Substrate Cofactor | 临用前配制，将Assay Buffer 全部加入到Substrate Cofactor中充分溶解，建议37℃水浴5min促进溶解 | 未用完的Working Substrate Cofactor 4℃保存1周 |
| 工作液 | 按Working Substrate：Working Substrate Cofactor=1:1的比例配制 | |
| Standard | 临用前配制，加1.71 mL去离子水，充分溶解得到40 μmol/mL标准品 | 未用完的Standard可4℃避光保存1周。若需长期保存，请分装后-20℃避光保存，避免反复冻融 |
注意：Chromogen和Substrate有一定的刺激性，建议在使用过程中做好个人防护。
2、标准曲线设置
按下表所示用去离子水将40 μmol/mL标准品稀释为8、4、2、1、0.5、0.25、0.125 μmol/mL的标准溶液。
| 序号 | 标准品体积 | 去离子水体积（µL） | 标准品浓度（μmol/mL） |
| Std.1 | 40µL of 40 μmol/mL | 160 | 8 |
| Std.2 | 100µL of Std.1 | 100 | 4 |
| Std.3 | 100µL of Std.2 | 100 | 2 |
| Std.4 | 100µL of Std.3 | 100 | 1 |
| Std.5 | 100µL of Std.4 | 100 | 0.5 |
| Std.6 | 100µL of Std.5 | 100 | 0.25 |
| Std.7 | 100µL of Std.6 | 100 | 0.125 |
注意：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。
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0.1g样本，加入1mL Extraction Buffer，室温下充分匀浆，转移到1.5mL EP管中，盖紧后（防止水分散失） 置于80℃水浴提取20min；冷却后，10,000g，室温离心10min，取上清液，待测。
3.2 植物组织
称取约0.1g样本，加入1mL Extraction Buffer捣碎，室温超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），转移到1.5mL EP管中，盖紧后（防止水分散失）置于80℃水浴提取20min；冷却后，10,000g，室温离心10min，取上清液，待测。
3.3 细胞或细菌
收集500万细胞或细菌到离心管内，用冷PBS清洗细胞，离心后弃上清，加入1mL Extraction Buffer，室温超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），转移到1.5mL EP管中，盖紧后（防止水分散失）置于80℃水浴提取20min；冷却后，10,000g，室温离心10min，取上清液，待测。
3.4 细胞上清或血清（浆）
在1.5mL EP管中，吸取0.5mL液体加入0.5mL Extraction Buffer，盖紧后（防止水分散失）置于80℃水浴提取20min；冷却后，10,000g，室温离心10min，取上清液，待测。
注意：推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒进行蛋白质浓度测定。
4、实验步骤
4.1 酶标仪或可见分光光度计预热30min以上，调节波长到570nm，可见分光光度计用去离子水调零。
4.2 样本测定（在EP管中依次加入下列试剂）：
| 试剂（μL） | 空白管 | 标准管 | 测定管 |
| 去离子水 | 10 | 0 | 0 |
| 不同浓度Std. | 0 | 10 | 0 |
| 样本 | 0 | 0 | 10 |
| 工作液 | 20 | 20 | 20 |
4.3 混匀后盖紧瓶盖（防止水分散失），置于沸水浴中保温5min，自来水冷却10s后加入170μL 60%乙醇反复颠倒EP管数次，5,000g 室温离心5min，每管取出150μL加入96孔板或微量玻璃比色皿，于570nm测定吸光值，计算ΔA测=A测定-A空白、ΔA标=A标准-A空白（空白管只需做1管）。显色后务必在30min内测完。
注意：空白管和标准管只需做1-2次。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测小于0.002可适当加大样本量。如果ΔA测大于1.8，样本可用去离子水进一步稀释，计算结果乘以稀释倍数。
5、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。
5.1 标准曲线的绘制
以标准液浓度为y轴，ΔA标为x轴绘制标准曲线。将ΔA测带入标准曲线公式计算出y（μmol/mL）。
5.2 样本赖氨酸含量计算
(1) 按样本质量计算
赖氨酸含量 (μmol/g 质量)=y÷(W÷V提取)×n=y÷W×n
(2) 按蛋白浓度计算
赖氨酸含量 (μmol/mg prot)=y÷Cpr×n
(3) 按细胞或细菌数量计算
赖氨酸(μmol/104 )=y÷(N÷ V提取)×n=y÷N ×n=y×n÷N
N：细胞或细菌数量，以10⁴ 为单位（例如细胞数量为5 ×10⁶，N=500）
(4) 按液体体积计算
赖氨酸含量 (μmol/mL)=y×2×n
参数说明：
W：样本质量，g；
V提取：加入Extraction Buffer体积，1mL；
n：样本进一步稀释的稀释倍数；
Cpr：上清液蛋白质浓度，mg/mL；
500：细菌或细胞总数，500万个；
2：提取液体时的稀释倍数，（0.5mL+0.5mL）/0.5mL=2。
6、结果展示
典型标准曲线
图1：赖氨酸含量标准曲线
实验实例
图2：本试剂盒测定小鼠肝脏、拟南芥中的赖氨酸含量
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。
FAQ
检测得到的样本 ΔA测过高或者过低怎么办？
如果 ΔA测小于 0.002，可适当加大样本量。如果 ΔA测大于 1.8，样本可用去离子水进一步稀释，计算结果乘以稀释倍数。
Lysine is one of the essential amino acids for the human body. It promotes development, enhances immune function, and improves the function of the central nervous system. Lysine is a basic essential amino acid. As cereal-based foods are very low in lysine content, which is easily destroyed and becomes deficient during processing, it is referred to as the first limiting amino acid.
Detection Principle:
The lysine in proteins possesses a free ε-NH₂ group, which reacts with the ninhydrin reagent to produce a blue-purple substance with a characteristic absorption peak at 570 nm. The lysine content is calculated by measuring the absorbance at 570 nm.
Detection Range: 0.125 - 8 µmol/mL
Sensitivity: 0.03125 µmol/mL
Applicable Samples: Serum; Plasma; Animal/Plant Tissues; Cells; Cell Supernatant; Bacteria
Note: It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before formal detection.
Reagents, consumables and Equipments not provided
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 570 nm)
96-well plate or micro glass cuvettes, adjustable pipettes and tips
Centrifuge, water bath
Deionized water, 95% ethanol, 60% ethanol
Homogenizer (for tissue samples)
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. |
| Working Substrate | Prepare immediately before use. For 96T, add 2 mL of 95% ethanol and dissolve completely. | Unused portion can be stored at 4°C protected from light for 1 week. |
| Working Substrate Cofactor | Prepare immediately before use. Add all of the Assay Buffer to the Substrate Cofactor and dissolve completely. A 5-minute incubation in a 37°C water bath is recommended to facilitate dissolution. | Unused portion can be stored at 4°C for 1 week. |
| Working Solution | Prepare by mixing Working Substrate and Working Substrate Cofactor at a 1:1 ratio. | |
| Standard | Prepare immediately before use. Add 1.71 mL of deionized water and dissolve completely to obtain a 40 µmol/mL standard solution. | Unused portion can be stored at 4°C protected from light for 1 week. For long-term storage, aliquot and store at -20°C protected from light, avoiding repeated freeze-thaw cycles. |
Note: Chromogen and Substrate can be irritating. Personal protection is recommended during use.
2. Standard Curve Setup
Dilute the 40 µmol/mL standard with deionized water as shown in the table below to prepare standard solutions of 8, 4, 2, 1, 0.5, 0.25, and 0.125 µmol/mL.
| Std. No. | Standard Volume | Deionized Water Volume (µL) | Standard Concentration (µmol/mL) |
| Std.1 | 40µL of 40 μmol/mL | 160 | 8 |
| Std.2 | 100µL of Std.1 | 100 | 4 |
| Std.3 | 100µL of Std.2 | 100 | 2 |
| Std.4 | 100µL of Std.3 | 100 | 1 |
| Std.5 | 100µL of Std.4 | 100 | 0.5 |
| Std.6 | 100µL of Std.5 | 100 | 0.25 |
| Std.7 | 100µL of Std.6 | 100 | 0.125 |
Note: A standard curve must be prepared 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 1 month.
3.1 Animal Tissue: Weigh about 0.1g of sample, add 1 mL of Extraction Buffer, homogenize thoroughly at room temperature. Transfer to a 1.5 mL EP tube, cap tightly (to prevent moisture loss), and incubate in an 80°C water bath for 20 min for extraction. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant for assay.
3.2 Plant Tissue: Weigh about 0.1g of sample, add 1 mL of Extraction Buffer and crush. Sonicate at room temperature for 5 min (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL EP tube, cap tightly, and incubate in an 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.
3.3 Cells or Bacteria: Collect 5 million cells/bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, discard supernatant. Add 1 mL of Extraction Buffer, sonicate as in 3.2. Transfer to EP tube, cap tightly, and incubate in 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.
3.4 Cell Supernatant or Serum/Plasma: In a 1.5 mL EP tube, add 0.5 mL of sample to 0.5 mL of Extraction Buffer. Cap tightly and incubate in an 80°C water bath for 20 min. After cooling, centrifuge at 10,000 g, room temperature for 10 min. Collect the supernatant.
Note: Protein concentration determination using the Aladdin BCA Protein Assay Kit (B665595) or Ready-to-Use BCA Protein Assay Kit (R1491648) is recommended.
4. Assay Procedure
4.1 Preheat the microplate reader or spectrophotometer for at least 30 min. Set wavelength to 570 nm. Zero the spectrophotometer with deionized water.
4.2 Sample Assay (Add reagents in the following order in EP tubes):
| Reagent (µL) | Blank Tube | Standard Tube | Assay Tube |
| Deionized Water | 10 | 0 | 0 |
| Different Conc. Std. | 0 | 10 | 0 |
| Sample | 0 | 0 | 10 |
| Working Solution | 20 | 20 | 20 |
4.3 Mix well, cap tubes tightly (prevent moisture loss), and place in a boiling water bath for 5 min. Cool under tap water for 10 seconds. Add 170 µL of 60% ethanol, invert tubes several times. Centrifuge at 5,000 g, room temperature for 5 min. Transfer 150 µL from each tube to a 96-well plate or micro cuvette. Measure absorbance at 570 nm. Calculate ΔAassay = Aassay - Ablank, ΔAstd = Astandard - Ablank (only 1 blank tube is needed). Measurement must be completed within 30 min after color development.
Note: Only 1-2 replicates of Blank and Standard tubes are needed. Preliminary tests with 2-3 samples of expected large differences are recommended before the formal experiment. If ΔAassay is less than 0.002, increase sample volume appropriately. If ΔAassay is greater than 1.8, further dilute the sample with deionized water and multiply the result by the dilution factor.
5. Calculation of Results
Note: Two formula formats are provided: a detailed derivation and a concise version. They are equivalent. The bold concise formula is recommended for final calculation.
5.1 Standard Curve Plotting: Plot standard concentration (y-axis) against ΔAstd (x-axis) to obtain the standard curve equation. Substitute ΔAassay into the equation to calculate y (µmol/mL).
5.2 Sample Lysine Content Calculation:
(1) Calculation Based on Sample Weight
Lysine Content (μmol/g weight) = y ÷ (W ÷ V<sub>Extraction</sub>) × n = y ÷ W × n
(2) Calculation Based on Protein Concentration
Lysine Content (μmol/mg protein) = y ÷ Cpr × n
(3) Calculation Based on Bacterial or Cell Count
Lysamine (μmol/104)=y÷(N÷Vextract)×n=y÷N×n=y×n÷N
N: number of cells or bacteria, in units of 104 (e.g., if the cell count is 5 × 106, N = 500).
(4) Calculation Based on Liquid Volume
Lysine Content (μmol/mL) = y × 2 × n
Parameter Definitions:
W: Sample weight (g)
Vextraction: Volume of Extraction Buffer added (1 mL)
n: Sample dilution factor (if further diluted)
Cpr: Protein concentration of the supernatant (mg/mL)
500: Total number of bacteria/cells (5 million)
2: Dilution factor for liquid samples, (0.5 mL + 0.5 mL) / 0.5 mL = 2
6. Result Presentation
Typical Standard Curve:
Figure 1: Standard curve for lysine content
Experimental Example:
Figure 2: Lysine content in mouse liver and Arabidopsis thaliana determined using this kit
Precautions
This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
FAQ
What should be done if the sample ΔAtest obtained from detection is too high or too low?
If ΔAtest is less than 0.002, appropriately increase the sample amount. If ΔAtest is greater than 1.8, further dilute the sample with deionized water, and multiply the calculated result by the dilution factor.
| L1509156 | Component | 96T | Storage |
| L1509156A | Extraction Buffer | 120 mL | 2-8℃ |
| L1509156B | Assay Buffer | 2 mL | 2-8℃ |
| L1509156C | Substrate | 1EA | 2-8℃. Store in the dark. |
| L1509156D | Substrate Cofactor | 1EA | 2-8℃. Store in the dark. |
| L1509156E | Standard | 1EA | 2-8℃. Store in the dark. |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0333023 |
分析证书 | L1509156 |
危险品化学品经营许可证（带存储）