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。
在加热和强酸条件下，尿素与二乙酰一肟及安替比林反应呈黄色，该黄色物质在波长 460nm 处有特征吸收峰。通过检测 460nm 处黄色物质吸光度的增加量，可计算得出样本中尿素的含量。
自备仪器和试剂
操作步骤（仅供参考）
建议先选取 1-3 个差异显著的样本（如不同类型 / 分组样本）进行预实验，熟悉操作流程后，根据预实验结果确定 / 调整样本稀释浓度，避免样本或试剂浪费。
1. 试剂准备
| 试剂名称 | 注意事项 |
| 试剂一 | 1. 开盖前需将粉体甩至管底（可手动轻甩）； 2. 加入 3mL 蒸馏水充分溶解； 3. 溶解后保存周期与试剂盒有效期一致。 |
| 试剂二 | 1. 开盖前需将粉体甩至管底（可手动轻甩）； 2. 先加入 2.4mL 蒸馏水溶解； 3. 缓慢加入 2.4mL 磷酸并充分混匀； 4. 再加入 4.8mL 硫酸混匀，冷却至室温后备用； 5. 配制后保存周期与试剂盒有效期一致。 |
| 标准管 | 每支标准管操作如下： 1. 临用前加入 1mL 蒸馏水溶解（浓度为 4mg/mL）； 2. 用蒸馏水按 1:39 比例稀释 40 倍，配制成 0.1mg/mL 的标准品； 3. 稀释后保存周期与试剂盒有效期一致。 |
2. 样本提取
2.1 组织样本
取 0.1g 组织样本，加入 1mL 蒸馏水研磨；超声萃取 10min 后定容至 1mL，室温静置 30min；12000rpm 常温离心 10min，取上清液待测。
2.2 液体样品
澄清液体可直接检测；若样品浑浊，需离心后取上清液检测。
3. 检测步骤
| 试剂组分 | 测定管 | 对照管 | 空白管（仅做一次） | 标准管（仅做一次） |
| 样本 | 50 | 50 | — | — |
| 蒸馏水 | — | 20 | 50 | — |
| 标准品 | — | — | — | 50 |
| 试剂一 | 20 | — | 20 | 20 |
| 试剂二 | 40 | 40 | 40 | 40 |
4. 结果计算
4.1 按组织质量计算
尿素含量 (mg/g) = (C标准×V1)×△A÷(A标准-A空白)÷(V1÷V×W)×D= 0.1×△A÷(A标准-A空白)÷W×D
尿素含量 (mg/kg) = (C标准×V1)×10³×△A÷(A标准-A空白)÷(V1÷V×W)×D = 100×△A÷(A标准-A空白)÷W×D
尿素氮含量 (μg/g) = (C标准×V1)×△A÷(A标准-A空白)÷(V1÷V×W)×D×10³÷60.04×2×14 = 46.64×△A÷(A标准-A空白)÷W×D
4.2 按液体体积计算
尿素含量 (mg/mL) = (C标准×V1)×△A÷(A标准-A空白)÷V1×D = 0.1×△A÷(A标准-A空白)×D
尿素氮含量 (mg/dL) = (C标准×V1)×△A÷(A标准-A空白)÷V1×D×100÷60.04×2×14 = 4.664×△A÷(A标准-A空白)×D
4.3 按蛋白浓度计算
尿素含量 (mg/mgprot) = (C标准×V1)×△A÷(A标准-A空白)÷(V1÷V×Cpr)×D = 0.1×△A÷(A标准-A空白)÷Cpr×D
尿素含量 (mg/mgprot) = (C标准×V1)×10³×△A÷(A标准-A空白)÷(V1÷V×Cpr)×D = 100×△A÷(A标准-A空白)÷Cpr×D
尿素氮含量 (μg/mgprot) = (C标准×V1)×△A÷(A标准-A空白)÷(V1÷V×Cpr)×D×10³÷60.04×2×14 = 46.64×△A÷(A标准-A空白)÷Cpr×D
公式参数说明
C标准：尿素标品浓度，0.1 mg/mL；
D：样本稀释倍数，未稀释时 D=1；
60.04：尿素分子量；
2：1 分子尿素含 2 个氮原子；
14：氮元素分子量；
W：取样组织质量（g）；
V：样本提取液总体积（1mL）；
V1：加入反应体系的样本体积（0.05mL）；
Cpr：样本蛋白浓度（mg/mL），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。
Under heating and strong acid conditions, urea reacts with diacetyl monoxime and antipyrine to form a yellow compound, which has a characteristic absorption peak at 460 nm. By measuring the increase in absorbance of the yellow compound at 460 nm, the urea content in the sample can be calculated.
Reagents, consumables and Equipments not provided
Microplate reader (capable of measuring absorbance at 460 nm)
Mortar (homogenizer), ice box (ice machine), benchtop centrifuge, water bath (oven, incubator, metal bath)
Adjustable pipettes, 96-well plates, centrifuge tubes
Distilled water (deionized water / ultrapure water are both acceptable)
Procedure (For Reference Only)
It is recommended to first select 1-3 samples with significant differences (e.g., different types/grouped samples) for a pilot experiment to familiarize yourself with the procedure. Based on the pilot experiment results, determine/adjust the sample dilution factor to avoid wasting samples or reagents.
1. Reagent Preparation
| Reagent | Instructions |
|---|---|
| Reagent A | 1. Before opening, tap the tube to ensure the powder settles at the bottom (can be done manually). 2. Add 3 mL of distilled water and dissolve completely. 3. After dissolution, the storage period is consistent with the kit's expiry date. |
| Reagent B | 1. Before opening, tap the tube to ensure the powder settles at the bottom (can be done manually). 2. First, add 2.4 mL of distilled water to dissolve. 3. Slowly add 2.4 mL of phosphoric acid and mix thoroughly. 4. Then add 4.8 mL of sulfuric acid, mix well, and cool to room temperature before use. 5. After preparation, the storage period is consistent with the kit's expiry date. |
| Standard Tube | For each standard tube, proceed as follows: 1. Before use, add 1 mL of distilled water to dissolve (concentration = 4 mg/mL). 2. Dilute 40-fold with distilled water at a 1:39 ratio to prepare a 0.1 mg/mL standard solution. 3. After dilution, the storage period is consistent with the kit's expiry date. |
2. Sample Preparation
2.1 Tissue Samples
Take 0.1 g of tissue sample and add 1 mL of distilled water. Homogenize.
Perform ultrasonic extraction for 10 min, then adjust the volume back to 1 mL. Let stand at room temperature for 30 min.
Centrifuge at 12,000 rpm at room temperature for 10 min. Collect the supernatant for testing.
2.2 Liquid Samples
Clear liquid samples can be tested directly.
If the sample is turbid, centrifuge and collect the supernatant for testing.
3. Assay Procedure
3.1 Pre-heat the microplate reader for 30 min, set the temperature to 37°C, and the detection wavelength to 460 nm.
3.2 Before the experiment, select 2 samples to determine the appropriate dilution factor (D) for this assay.
3.3 Thaw all reagents to room temperature. Add the following volumes (in µL) to EP tubes:
| Reagent Component | Test Tube | Control Tube | Blank Tube (Perform once) | Standard Tube (Perform once) |
|---|---|---|---|---|
| Sample | 50 | 50 | — | — |
| Distilled Water | — | 20 | 50 | — |
| Standard Solution | — | — | — | 50 |
| Reagent A | 20 | — | 20 | 20 |
| Reagent B | 40 | 40 | 40 | 40 |
3.4 Mix thoroughly. Incubate in a 95°C boiling water bath for 20 min.
3.5 Add 190 µL of distilled water to each tube and mix again. If the solution becomes turbid, centrifuge at 5,000 rpm at room temperature for 5 min and collect the supernatant.
3.6 Transfer 200 µL of the above solution to a 96-well plate. Measure the absorbance (A) at 460 nm. Calculate ∆A = A<sub>Test</sub> - A<sub>Control</sub>.
【Note】 If the absorbance (A) of the Test Tube exceeds 1.5, further dilute the sample and incorporate the final dilution factor (D) into the subsequent calculation formula.
4. Calculation of Results
4.1 Calculation Based on Tissue Weight
Urea Content (mg/g tissue) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D
= 0.1 × ∆A ÷ (AStandard - ABlank) ÷ W × D
Urea Content (mg/kg tissue) = (CStandard × V1) × 10³ × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D
= 100 × ∆A ÷ (AStandard - ABlank) ÷ W × D
Urea Nitrogen Content (µg/g tissue) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × W) × D × 10³ ÷ 60.04 × 2 × 14
= 46.64 × ∆A ÷ (AStandard - ABlank) ÷ W × D
4.2 Calculation Based on Liquid Volume
Urea Content (mg/mL) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ V1 × D
= 0.1 × ∆A ÷ (AStandard - ABlank) × D
Urea Nitrogen Content (mg/dL) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ V1 × D × 100 ÷ 60.04 × 2 × 14
= 4.664 × ∆A ÷ (AStandard - ABlank) × D
4.3 Calculation Based on Protein Concentration
Urea Content (mg/mg prot) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D
= 0.1 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Urea Content (mg/g prot) = (CStandard × V1) × 10³ × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D
= 100 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Urea Nitrogen Content (µg/mg prot) = (CStandard × V1) × ∆A ÷ (AStandard - ABlank) ÷ (V1 ÷ V × Cpr) × D × 10³ ÷ 60.04 × 2 × 14
= 46.64 × ∆A ÷ (AStandard - ABlank) ÷ Cpr × D
Parameter Description for Formulas
C<sub>Standard</sub>: Concentration of the urea standard, 0.1 mg/mL.
D: Sample dilution factor. D=1 if the sample is undiluted.
60.04: Molecular weight of urea.
2: One molecule of urea contains two nitrogen atoms.
14: Atomic weight of nitrogen.
W: Weight of the tissue sample taken (g).
V: Total volume of the sample extract (1 mL).
V<sub>1</sub>: Volume of the sample added to the reaction system (0.05 mL).
C<sub>pr</sub>: Protein concentration of the sample (mg/mL). It is recommended to use Aladdin's B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.
Precautions
Before formal testing, it is recommended to select 2-3 samples with expected significant differences for a pilot experiment.
For tissue samples and cell samples, results can be normalized between samples by measuring the protein concentration. It is recommended to use Aladdin's B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.
Biochemical detection reagents are generally irritating and have biological toxicity. For your safety and health, please ensure proper biosafety protection throughout the experiment. Wear a lab coat, mask, gloves, head cover, and other safety gear. Conduct experiments in a fume hood or biosafety cabinet.
This product is for scientific research only and is not intended for clinical diagnosis.
| U1516000 | Component | 96T | Storage |
| U1516000A | 试剂一 | 1 EA | 2-8℃. |
| U1516000B | 试剂二 | 1 EA | 2-8℃. |
| U1516000C | 标准管 | 1 EA×2 | 2-8℃. |
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 |
|---|---|---|
 ZJ26F0838266 |
分析证书 | U1516000 |
| ZJ26F0434006 |
分析证书 | U1516000 |
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