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。
脲酶（UE，EC 3.5.1.5）能够水解尿素，产生氨和碳酸。UE 活性与有机物质含量、全氮和速效氮含量呈正相关，反映了氮素状况。
本试剂盒检测原理是利用靛酚蓝比色法测定脲酶水解尿素产生的 NH₃-N，在 630 nm 处有特征吸收峰。
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注意：
1.请在实验前检查各组分的量。
2.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
酶标仪，96 孔酶标板（普通透明板，PBS，去离子水，匀浆器（组织样本），恒温箱，低温离心机，可调节式移液枪及枪头
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。
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.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。
实验流程
1 试剂准备
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2 标准品配制
用提取缓冲液将 1 M NH₄Cl 标准品稀释成 1 mM，使用 1 mM 标准液，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。
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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。
动植物组织：称取约 0.1 g 组织样本，加入 1 mL 提取缓冲液，冰浴匀浆，8000 g，4℃离心 10 min，
取上清，置冰上待测。
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提取缓冲液，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），8000 g，4℃离心 10 min，取上清，置冰上待测。
血清（浆）：直接测定。
4 实验步骤
（1）酶标仪准备：预热 30 min 以上，调节波长到 630 nm。
（2）酶促反应：在 1.5 mL EP 管中按照如下方式操作：
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（3）混匀，放入 37℃水浴 1 h 后，10000 g，25℃离心 10 min，取上清液。将上清液稀释 10 倍（取 0.1 mL 上清液，加入 0.9 mL 去离子水）。
（4）测氨量：在 96 孔板中按照如下方式操作：（空白孔和标准孔只需测定 1 次）
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（5） 吸光度检测：快速混匀，在 630 纳米波长下测定吸光度，分别记录吸光值为 A测定、A对照、A标准、 A空白
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测定 = A测定 − A对照，ΔA标准 = A标准 − A空白
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（μM）。
3. UE 活性的计算:
● 按样本质量计算:
● 按样本蛋白浓度计算:
单位的定义：每 g 组织每分钟产生 1 μmol 的 NH₃-N 定义为一个酶活力单位。
UE (U/g)=y×(V反总÷V样)×10×V样总÷T÷1000÷W=0.0025y÷W
单位的定义：每 mg 组织蛋白每分钟产生 1 μmol 的 NH₃-N 定义为一个酶活力单位。
UE (U/mg prot)=y×(V反总÷V样)×10÷T÷1000÷Cpr=0.0025y÷Cpr
● 按细胞或细菌数量计算:
单位的定义：每 10⁶ 个细胞或细菌每分钟产生 1 μmol 的 NH₃-N 定义为一个酶活力单位。
UE (U/10⁶)=y×(V反总÷V样)×10×V样总÷T÷1000÷N=0.0025y÷N
● 按血清（浆）体积计算:
单位的定义：每 mL 血清（浆）每分钟产生 1 μmol 的 NH₃-N 定义为一个酶活力单位。
UE (U/mL)=y×(V反总÷V样)×10÷T÷1000=0.0025y
V反总：反应体系总体积，0.3 mL；V样：加入样本体积，0.02 mL；T：反应时间，60 min；1000：单位换算系数，1 L=1000 mL；Cpr：蛋白浓度，mg/mL；10：稀释倍数；V样总：提取缓冲液体积，1 mL；W：样本质量，g；N：细胞或细菌数量，以 10⁶ 为单位（例如细菌数量为 5×10⁶，N=5）。
结果展示
典型标准曲线：
示例 - 1: 取 0.1 g 小鼠肝脏使用 96 孔板测得计算 ΔA测定 = 0.206−0.09=0.116，代入方程得 y=196.995，按样本质量计算得：UE (U/g)=0.0025×196.995÷0.1=4.924 U/g。
示例 - 2: 取 10⁶ Jurkat 细胞使用 96 孔板测得计算 ΔA测定 = 0.110−0.044=0.066，代入方程得 y=111.84，按细胞数量计算得：UE (U/10⁶)=0.0025×111.7753÷5=0.056 U/10⁶。
示例 - 3: 取 20 μL 牛血清使用 96 孔板测得计算 ΔA测定 = 0.210−0.093=0.117，代入方程得 y=198.65，按液体体积计算得：UE (U/mL)=0.0025×198.689=0.497 U/mL。
FAQ
检测得到的样本 ΔA测定 过高或者过低怎么办？
如果样本 ΔA测定 ＞0.6，样本需用提取缓冲液适当稀释或减少提取用样本量，重新进行测定。如果样本的 ΔA测定 ＜0.001，可适当提高样本量。
Urease (UE, EC 3.5.1.5) catalyzes the hydrolysis of urea to produce ammonia and carbonic acid. UE activity is positively correlated with organic matter content, total nitrogen and available nitrogen levels, serving as an indicator of soil and tissue nitrogen status.
This kit adopts indophenol blue colorimetry to quantify NH₃-N generated from urea hydrolysis by urease, with maximum absorbance at 630 nm.
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Notes：
1.Verify the volume of each kit component before starting experiments.
2.All reagents are supplied with an extra 10% volume on top of the standard specification for standard curve preparation and preliminary trial tests.
Self-supplied Instruments and Reagents
Microplate reader, transparent 96-well microplates, PBS, deionized water, tissue homogenizer (for tissue samples), constant temperature incubator, refrigerated centrifuge, adjustable pipettes and pipette tips
General Precautions
1.Conduct preliminary trials with 2–3 samples exhibiting expected large differences before formal detection.
2.For tissue, cell and bacterial samples, normalize final results by total protein concentration to eliminate inter-sample variation.
3.This kit is compatible with spectrophotometer detection; proportionally adjust reagent volumes following spectrophotometer operating requirements.
4.It is recommended to generate an independent standard curve for each experiment to improve detection accuracy. If no self-made standard curve is available, calculate results using the typical standard curve formula provided in the Results section.
5.Biochemical assay reagents are generally irritant and biohazardous. Wear full personal protective equipment including lab coat, mask, gloves and hairnet throughout all operations, and perform all experimental steps in a fume hood or biosafety cabinet for occupational safety.
6.This product is for research use only; not intended for clinical diagnostic applications.
Experimental Procedures
1. Reagent Preparation
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2. Standard Working Solution Preparation
Dilute 1 M NH₄Cl Standard with Extraction Solution to prepare a 1 mM stock standard. Further serially dilute the 1 mM standard solution as specified in the table below. Prepare and analyze a standard curve alongside every independent experiment. Diluted standard solutions are unstable and must be used within 4 hours of preparation.
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3. Sample Preparation
Notes: Fresh samples are recommended for immediate detection. If testing cannot be performed right away, samples may be stored at −80 °C for up to 1 month.
Animal & Plant Tissues: Weigh approximately 0.1 g tissue sample, add 1 mL Extraction Solution, homogenize on ice, centrifuge at 8000 g, 4 °C for 10 min. Collect the supernatant and keep on ice prior to assay.
Cells or Bacteria: Harvest 5×10⁶ cells/bacteria, wash with cold PBS, centrifuge at 800 g for 2 min and discard supernatant. Resuspend pellet in 1 mL Extraction Solution, lyse by sonication on ice for 5 min (20% power / 200 W, 3 s sonication / 7 s interval, repeat 30 cycles). Centrifuge lysate at 8000 g, 4 °C for 10 min, collect supernatant and store on ice.
Serum / Plasma: Detect directly without pre-processing.
4. Assay Protocol
(1) Microplate reader setup: Preheat the instrument for ≥30 min and set detection wavelength to 630 nm.
(2) Enzymatic reaction: Prepare reaction mixtures in 1.5 mL EP tubes as outlined below:
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(3) Thoroughly mix all tubes, incubate in a 37 °C water bath for 1 h, then centrifuge at 10000 g, 25 °C for 10 min. Collect supernatant and dilute 10-fold (mix 0.1 mL supernatant with 0.9 mL deionized water).
(4) Ammonia quantification: Pipette reagents into a 96-well plate following the plate layout below (blank and standard wells only require single measurement per run).
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(5) Absorbance measurement: Mix each well rapidly, read absorbance at 630 nm, record values asAsample, Acontrol, Astandard and Ablank respectively.
Calculation of Urease Activity
Two equivalent formulas are provided below: the full derivation formula and simplified computational formula.
1 Raw Data Calculation
2 Standard Curve Construction
ΔAsample = Asample − Acontrol
ΔAstandard = Astandard − Ablank
Plot standard curve with standard concentration as the y-axis and ΔAstandard as the x-axis. Substitute ΔAsample into the curve equation to obtain y value (unit: μM).
3 Urease Activity Calculation
● Calculation by Tissue Weight:
Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per gram of tissue per minute.
UE (U/g)=y×(Vtotal-reaction÷Vsample)×10×Vextract÷T÷1000÷W=0.0025y÷W
● Calculation by Total Protein Concentration:
Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per milligram of total protein per minute.
UE (U/mg prot)=y×(Vtotal-reaction÷Vsample)×10÷T÷1000÷Cpr=0.0025y÷Cpr
●Calculation by Cell/Bacterial Count::
Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH-N per 10⁶ cells or bacteria per minute.
UE (U/10⁶)=y×(Vtotal-reaction÷Vsample)×10×Vextract÷T÷1000÷N=0.0025y÷N
●Calculation by Serum/Plasma Volume:
Unit definition: One unit (U) refers to the amount of urease that produces 1 μmol NH₃-N per milliliter of serum/plasma per minute. T: Enzymatic reaction incubation time = 60 min
UE (U/mL)=y×(Vtotal-reaction÷Vsample)×10÷T÷1000=0.0025y
V<sub>total-reaction</sub>: Total volume of enzymatic reaction system = 0.3 mL
V<sub>sample</sub>: Volume of sample added to reaction = 0.02 mL
1000: Unit conversion factor (1 L = 1000 mL)
Cpr: Total protein concentration of sample (mg/mL)
10: 10-fold dilution factor for post-reaction supernatant
V<sub>extraction</sub>: Volume of Extraction Solution used for sample lysis = 1 mL
W: Weight of tissue sample (g)
N: Cell/bacterial count in units of 10⁶ (e.g., 5×10⁶ cells corresponds to N=5)
Representative Results
Typical Standard Curve:
Calculation Examples:
1.Tissue sample example: 0.1 g mouse liver tissue was assayed in 96-well plate.
ΔAsample = 0.206 − 0.09 = 0.116. Substitute into standard curve to get y = 196.995. UE (U/g) = 0.0025 × 196.995 ÷ 0.1 = 4.924 U/g
2.Cell sample example: 1×10⁶ Jurkat cells were assayed.
ΔAsample = 0.110 − 0.044 = 0.066. Substitute into standard curve to get y = 111.7753, N=5. UE (U/10⁶ cells) = 0.0025 × 111.7753 ÷ 5 = 0.056 U/10⁶
3.Serum example: 20μL bovine serum was assayed.
ΔAsample = 0.210 − 0.093 = 0.117. Substitute into standard curve to get y = 198.689. UE (U/mL serum) = 0.0025 × 198.689 = 0.497 U/mL
FAQ
What to do if measured ΔA<sub>sample</sub> is excessively high or low?
If ΔA<sub>sample</sub> > 0.6: Dilute the original sample with Extraction Solution or reduce the starting sample weight/cell number, then re-assay.
If ΔA<sub>sample</sub> < 0.001: Increase the mass/volume of starting sample appropriately before re-testing.
| 项目号 | 物理外观 | 组件 | 48T | 96T | 储存 |
| U1521990A | 液体 | 提取缓冲液 | 70 mL | 70 mL×2 | 2-8 ℃ |
| U1521990B | 粉末 | 试剂一 | 1瓶 | 1瓶 | 2-8 ℃ |
| U1521990C | 液体 | 试剂二 | 25 mL | 50 mL | 2-8 ℃ |
| U1521990D | 液体 | 试剂三A | 0.4 mL | 0.8 mL | 2-8 ℃ |
| U1521990E | 液体 | 试剂三B | 1.6 mL | 3.2 mL | 2-8 ℃ |
| U1521990F | 液体 | 试剂四 | 2 mL | 4 mL | 2-8 ℃. Store in the dark. |
| U1521990G | 液体 | 标准品 | 0.2 mL | 0.4 mL | 2-8 ℃ |
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