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。
酪氨酸酶（Tyrosinase，EC1.14.18.1）是一种结构复杂的多亚基的含铜氧化还原酶，广泛存在于微生物、动植物及人体内，是生物体合成黑色素的关键酶，也与果蔬褐变、真菌组织保护以及昆虫伤口愈合及发育等过程密切相关。
本试剂盒检测原理是酪氨酸酶能够催化L-多巴生成多巴醌，多巴醌能与MBTH生成粉色偶联物，在505nm处测定吸光值可计算出酪氨酸酶的活性。
组分与储存：
*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 (能测505nm处的吸光度)
* 96孔酶标板 (普通酶标板)
* PBS/去离子水 (样本清洗用/检测体系配制用)
* 匀浆器（组织样本）、恒温水浴锅、制冰机、低温离心机、可调节式移液枪及枪头 (检测量较大时合理使用多通道移液器有助于提高实验效率)
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。推荐使用阿拉丁货号：R1491648 的蛋白质定量试剂盒（BCA法）。
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. 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。
5. 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：
① 提取液：即用型；实验过程中冰上放置，2-8°C保存。
② 试剂一：临用前配制；48T加入12mL 提取液，96T加入24mL提取液，充分溶解待用。
注意：未用完的试剂分装后-20°C避光可保存4周，避免反复冻融；试剂一溶解慢，需要用移液枪充分吹打混匀，吹打过程中不要产生过多气泡。
③ 试剂二：即用型；使用前平衡到室温，2-8°C，避光保存。
④ 试剂三：即用型；使用前平衡到室温，2-8°C保存。
2. 样本制备：
注意：
① 推荐使用新鲜样本。样本尽量新鲜提取，如果不能当天提取，则把完整细胞或者分装的组织块保存在-80°C，一个月内使用。当准备好进行该项实验时，将样本从-80°C拿出后，冰上解冻。
② 样本处理完后吸取上清时不要吸取到沉淀和上层杂质（部分组织样本），如果吸取到可进行二次离心去除。
* 动植物组织：称取约0.1g组织样本，加入1mL提取液，使用预冷好的研磨仪冰浴匀浆（推荐参数：加入3颗3mm钢珠，55Hz，匀浆30s，间隔10s，重复4次），12000g，4°C离心10min，取上清液，置冰上待测。
* 细胞或细菌：收集5×10⁶个细胞或细菌到离心管内，用预冷的PBS清洗细胞，800g离心2min，弃上清。加入1mL提取液，使用预冷好的研磨仪冰浴匀浆（推荐参数：加入3颗3mm钢珠，40Hz，匀浆20s，间隔10s，重复3次），12000g，4°C离心10min，取上清液，置冰上待测。
* 真菌（如平菇等）：称取约0.1g样本，加入1mL提取液，冰浴匀浆或手动研磨至无明显组织块（推荐手磨），12000g，4°C离心10min，取上清液，置冰上待测。
* 血清（浆）：直接检测。
3. 实验步骤：
① 酶标仪准备：预热30min以上，调节波长到505nm。
② 检测体系配制：空白管需做1-2次。在1.5mLEP管中按照如下方式操作：
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③ 吸光度检测：充分混匀，12000g，4°C离心3min。取200μL上清至96孔板中，测定505nm处的吸光值，测定孔记为A测定，空白孔记为A空白。
结果计算：
1. 数据处理计算ΔA测定=A测定-A空白
2. 样本酪氨酸酶活性计算
* 按样本蛋白浓度计算：
单位的定义：每mg组织蛋白在反应体系中每分钟使505nm下的吸光值变化0.005定义为一个酶活单位。
酪氨酸酶活性(U/mg prot)=ΔA测定×V反总÷(V样×Cpr)÷0.005÷T=103.7×ΔA测定÷Cpr
* 按样本质量计算：
单位的定义：每g组织在反应体系中每分钟使505nm下的吸光值变化0.005定义为一个酶活单位。
酪氨酸酶活性 (U/g)=ΔA测定×V反总÷(V样÷V样总×W)÷0.005÷T=103.7×ΔA测定÷W
* 按细胞或细菌数量计算：
单位的定义：每10⁶个细胞或细菌在反应体系中每分钟使505nm下的吸光值变化0.005定义为一个酶活单位。
酪氨酸酶活性 (U/106)=ΔA测定×V反总÷(V样÷V样总×N)÷0.005÷T=103.7×ΔA测定÷N
* 按血清（浆）体积计算：
单位的定义：每mL血清（浆）在反应体系中每分钟使505nm下的吸光值变化0.005定义为一个酶活单位。酪氨酸酶活性 (U/mL)=ΔA测定×V反总÷V样÷0.005÷T=103.7×ΔA测定
V反总：反应体系总体积，0.28mL；
V样：加入反应体系中样本体积，0.027mL；
V样总：加入提取液体积，1mL；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
T：反应时间，20min；
N：细胞或细菌数量，以10⁶为单位（例如细胞数量为5×10⁶，N=5）。
结果展示：
示例1：取0.103g小鼠肾脏，按照测定步骤操作，用96孔板进行检测。
测得：A测定=0.143，A空白=0.048，ΔA测定=A测定-A空白=0.143-0.048=0.095。
按样本质量计算得：酪氨酸酶活性(U/g)=103.7×ΔA测定÷W=95.646U/g。
示例2：取0.100g菠菜，按照测定步骤操作，用96孔板进行检测。
测得：A测定=0.293，A空白=0.046，ΔA测定=A测定-A空白=0.293-0.046=0.247。
按样本质量计算得：酪氨酸酶活性(U/g)=103.7×ΔA测定÷W=256.139U/g。
常见问题：
检测得到的样本ΔA测定过高或者过低怎么办？
如果ΔA测定小于0.01可适当增加加入的样本体积或提高样本量。如果ΔA测定大于1，可将样本用提取液稀释，或减少加入的样本体积或降低样本量，再重新进行测定。
Tyrosinase (EC 1.14.18.1) is a structurally complex, multi-subunit copper-containing oxidoreductase. It is widely distributed in microorganisms, plants, animals and humans. As the key enzyme for melanin biosynthesis in organisms, it is also closely associated with fruit and vegetable browning, fungal tissue protection, as well as wound healing and development in insects.
The detection principle of this kit is as follows: tyrosinase catalyzes L-dopa to produce dopaquinone, which reacts with MBTH to form a pink conjugate. The absorbance value measured at 505 nm can be used to calculate tyrosinase activity.
Components and Storage:
*An extra 10% of each component is supplied on top of the stated specification for standard curve preparation or preliminary tests. |
Instruments and Reagents to Be Prepared by Users:
* Microplate reader (capable of measuring absorbance at 505 nm)
* 96-well microplate (standard type)
* PBS / deionized water (for sample washing and reaction system preparation)
* Homogenizer (for tissue samples), thermostatted water bath, ice maker, refrigerated centrifuge, adjustable pipettes and pipette tips
(For high-throughput testing, multi-channel pipettes are recommended to improve experimental efficiency)
Precautions:
1. Prior to formal detection, it is recommended to conduct preliminary tests with 2–3 samples expected to show significant differences.
2. For tissue and cell samples, protein concentration measurement is available to normalize results among different samples. BCA Protein Quantification Kit (Aladdin Cat. No. R1491648) is recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the volume of detection reagents proportionally in accordance with spectrophotometer operation requirements.
4. Biochemical test reagents are generally irritant and biologically toxic. For personal safety and health, complete biosafety protection must be implemented throughout the experiment, including lab coat, mask, gloves and hairnet. All operations shall be carried out in a fume hood or biosafety cabinet.
5. This product is for research use only and not intended for clinical diagnosis.
Experimental Procedure:
1. Reagent Preparation:
① Extraction Solution: Ready-to-use; keep on ice during experiments, store at 2–8°C.
② Reagent I: Prepare right before use. For 48-test kit: add 12 mL extraction solution; For 96-test kit: add 24 mL extraction solution. Mix completely until fully dissolved for subsequent use.
Notes: Aliquot leftover reagent and store at −20°C protected from light for up to 4 weeks; avoid repeated freeze-thaw cycles. Reagent I dissolves slowly. Thoroughly pipette up and down to mix, and prevent excessive bubbles during pipetting.
③ Reagent II: Ready-to-use; equilibrate to room temperature prior to use, store at 2–8°C away from light.
④ Reagent III: Ready-to-use; equilibrate to room temperature prior to use, store at 2–8°C.
2. Sample Preparation:
Notes:
① Fresh samples are recommended. Perform sample extraction as soon as possible after sampling. If extraction cannot be completed on the same day, store intact cells or aliquoted tissue pieces at −80°C and use within one month. Thaw samples on ice after taking them out of −80°C storage when ready for the assay.
② Avoid aspirating precipitates and floating impurities when collecting supernatant after sample processing (common in some tissue samples). Re-centrifuge the sample to remove contaminants if impurities are drawn.
* Animal and plant tissues: Weigh approximately 0.1 g tissue sample, add 1 mL extraction solution, and homogenize on ice with a pre-chilled tissue homogenizer.
Recommended parameters: add three 3 mm steel beads, 55 Hz, homogenize for 30 s, pause for 10 s, repeat 4 cycles.
Centrifuge at 12000 g and 4°C for 10 min, collect supernatant and keep on ice for testing.
* Cells or bacteria: Transfer 5×10⁶ cells or bacteria into a centrifuge tube, wash pellets with pre-chilled PBS, centrifuge at 800 g for 2 min, then discard supernatant. Add 1 mL extraction solution and homogenize on ice with a pre-chilled tissue homogenizer.
Recommended parameters: add three 3 mm steel beads, 40 Hz, homogenize for 20 s, pause for 10 s, repeat 3 cycles.
Centrifuge at 12000 g and 4°C for 10 min, collect supernatant and keep on ice for testing.
* Fungi (e.g., oyster mushroom): Weigh approximately 0.1 g sample, add 1 mL extraction solution, homogenize on ice or grind manually until no obvious tissue clumps remain (manual grinding preferred). Centrifuge at 12000 g and 4°C for 10 min, collect supernatant and keep on ice for testing.
* Serum / Plasma: Ready for direct detection without pretreatment.
3. Assay Steps:
① Microplate reader preparation: Preheat for more than 30 min and set the wavelength to 505 nm.
② Reaction system preparation: Prepare 1–2 blank tubes. Perform operations in 1.5 mL EP tubes as shown below:
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③ Absorbance measurement: Mix thoroughly, centrifuge at 12000 g and 4 °C for 3 min. Transfer 200 μL supernatant into a 96-well plate, then measure absorbance at 505 nm. Record the absorbance of test wells as A<sub>test</sub> and blank wells as A<sub>blank</sub>.
Result Calculation:
1. Data processing ΔAtest = Atest − Ablank
2. Calculation of sample tyrosinase activity
* Calculation based on sample protein concentration:
Unit definition: One unit (U) of enzyme activity is defined as the amount of enzyme that causes an absorbance change of 0.005 at 505 nm per minute per mg total tissue protein in the reaction system.
Tyrosinase activity (U/mg prot) = ΔAtest × Vtotal reaction ÷ (Vsample × Cpr) ÷ 0.005 ÷ T = 103.7 × ΔAtest ÷ Cpr
* Calculation based on sample weight:
Unit definition: One unit (U) of enzyme activity is defined as the amount of enzyme that causes an absorbance change of 0.005 at 505 nm per minute per gram of tissue in the reaction system.
Tyrosinase activity (U/g) = ΔAtest × Vtotal reaction ÷ (Vsample ÷ Vtotal sample extract × W) ÷ 0.005 ÷ T = 103.7 × ΔAtest ÷ W
* Calculation based on cell or bacterial count:
Unit definition: One unit (U) of enzyme activity is defined as the amount of enzyme that causes an absorbance change of 0.005 at 505 nm per minute per 10⁶ cells or bacteria in the reaction system.
Tyrosinase activity (U/10⁶ cells) = ΔAtest × Vtotal reaction ÷ (Vsample ÷ Vtotal sample extract × N) ÷ 0.005 ÷ T = 103.7 × ΔAtest ÷ N
* Calculation based on serum/plasma volume:
Unit definition: One unit (U) of enzyme activity is defined as the amount of enzyme that causes an absorbance change of 0.005 at 505 nm per minute per mL serum or plasma in the reaction system.
Tyrosinase activity (U/mL) = ΔAtest × Vtotal reaction ÷ Vsample ÷ 0.005 ÷ T = 103.7 × ΔAtest
V<sub>total reaction</sub>: Total volume of reaction system, 0.28 mL
V<sub>sample</sub>: Volume of sample added to the reaction system, 0.027 mL
V<sub>total extract</sub>: Total volume of extraction solution added to raw sample, 1 mL
Cpr: Protein concentration of sample extract, mg/mL
W: Weight of raw sample, g
T: Reaction incubation time, 20 min
N: Number of cells or bacteria, in units of 10⁶ (e.g., if total cell count is 5×10⁶, N = 5)
Result Presentation:
Example 1
0.103 g mouse kidney tissue was collected and assayed following the standard protocol with a 96-well plate.
Measured values: Atest = 0.143, Ablank = 0.048
ΔAtest = Atest − Ablank = 0.143 − 0.048 = 0.095
Calculated by sample weight: Tyrosinase activity (U/g) = 103.7 × ΔA<sub>test</sub> ÷ W = 95.646 U/g
Example 2
0.100 g spinach tissue was collected and assayed following the standard protocol with a 96-well plate.
Measured values: Atest = 0.293, Ablank = 0.046
ΔAtest = Atest − Ablank = 0.293 − 0.046 = 0.247
Calculated by sample weight: Tyrosinase activity (U/g) = 103.7 × ΔA<sub>test</sub> ÷ W = 256.139 U/g
Troubleshooting:
What to do if the measured ΔA<sub>test</sub> is excessively high or low?
If ΔA<sub>test</sub> is less than 0.01, appropriately increase the volume of sample added or raise the starting sample amount.
If ΔA<sub>test</sub> exceeds 1, dilute the sample with extraction solution, reduce the volume of sample added or lower the starting sample amount, then repeat the assay.
| 项目号 | 物理外观 | 组件 | 48T | 96T | 储存 |
| T1524567A | 液体 | 提取液 | 72 mL | 72 mL×2 | 2-8℃. |
| T1524567B | 固体 | 试剂一 | 1 vial | 1 vial | 2-8℃. Store in the dark. |
| T1524567C | 液体 | 试剂二 | 2.4 mL | 4.8 mL | 2-8℃. Store in the dark. |
| T1524567D | 液体 | 试剂三 | 0.9 mL | 1.8 mL | 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 |
|---|---|---|
 ZJ26F0939455 |
分析证书 | T1524567 |
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