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。
细胞色素 P450 酶超家族是一类广泛存在于生物体内的含血红素的单加氧酶，在药物代谢、外源化合物解毒及内源性物质合成中发挥关键作用。其最显著的特性包括底物广谱性、催化反应多样性（如羟化、脱烷基、环氧化等）以及依赖还原型辅酶Ⅱ（NADPH）和分子氧的电子传递链。不同亚型的 P450 酶具有独特的组织分布与底物选择性，常被用作评估药物相互作用及环境污染物毒性的生物标志物。
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实验流程
试剂准备
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1-2 标准品配制
取 10 μL 0.25 mM 标准品，加入 90 μL 提取缓冲液混匀稀释，即得 25 nmol/mL 标准品，按下表所示，进行下一步稀释。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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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℃，一个月内使用。当准备好进行该项实验时，将样本从-80℃拿出后，冰上解冻。但是请注意，这可能会影响样本的稳定性，实验结果可能会低于预期。
2. 肝脏样本中富含的 P450 酶活较高，可将上清液用提取缓冲液稀释至 20-40 倍后再进行检测。
● 动物组织：称取约 0.1 g 组织样本，加入1 mL提取缓冲液，冰浴匀浆，10000 g，4℃离心 10 min，取上清液待测。
3 实验步骤
1. 酶标仪准备：预热 30 min 以上，调节激发波长为 380 nm，发射波长为 450 nm。
2. 工作试剂：将试剂三与提取缓冲液进行 1：4 混匀，现用现配。
3. 工作液的配制：
标品工作液：每个标准孔/空白孔准备 100 µL，现配现用：吸取 90 µL试剂一，10 µL 试剂二，配制好的工作液需避光放置，使用前需混匀。
测定工作液：每个测定孔准备 100 µL 测定工作液，现配现用：吸取 80 µL试剂一，10 µL 试剂二，10 µL工作试剂，配制好的工作液需避光放置，使用前需混匀。
对照工作液：每个对照孔准备 100 µL 对照工作液，现配现用：吸取 80 µL试剂一，10 µL 去离子水，10 µL工作试剂，配制好的工作液需避光放置，使用前需混匀。
4. 检测体系配制：每个测定孔需设一个对照孔，空白孔和标准孔只用测 1 次。实验操作需要在避光环境下进行。在全黑 96 孔板中按照如下方式操作：
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5. 荧光检测：混匀后，记录各孔的荧光值 RFU，荧光激发波长 380 nm，荧光发射波长 450 nm，测定孔记为 RFU测定，对照孔记为 RFU对照，空白孔记为 RFU空白，标准孔记为 RFU标准。
结果计算
1. 数据处理
计算ΔRFU测定 = RFU测定 − RFU对照，ΔRFU标准 = RFU标准 − RFU空白
2.标准曲线绘制
以标准溶液浓度为 y 轴，ΔRFU标准 为 x 轴，绘制标准曲线，得到标准方程，将 ΔRFU测定 带入方程得到 y (nmol/mL)。
3. 样本 P450 酶活性计算
● 按样本蛋白浓度计算：单位定义：37℃下每 mg 蛋白每分钟催化产生 1 nmol 的 7-羟基香豆素为一个酶活力单位。
P450 酶活性 (U/mg prot) = V样 × y ÷ (V样 × Cpr) ÷ T = y ÷ Cpr ÷ 10
● 按样本质量计算： 单位定义：37℃下每 g 组织每分钟催化产生 1 nmol 的 7-羟基香豆素为一个酶活力单位。
P450 酶活性 (U/g) = V样总 × y ÷ W ÷ T = y ÷ W ÷ 10
V样总：提取液总量，1 mL；
T：反应时间，10 min；W：样本质量，g；
V样：加入反应体系中上清液体积，0.02 mL
结果展示
典型标准曲线：y=0.0001x-0.0009， R2=0.9988
示例-1: 取 0.1019 g 兔肝，用提取缓冲液稀释 20 倍，按照测定步骤操作，用全黑 96 孔板测得：ΔRFU测定=RFU测定−RFU对照=18983−6787=12196，代入标准曲线，计算 y=1.219。按样本质量计算得：P450酶活性 (U/g)=y÷W÷10×20=23.925 U/g。
FAQ
● 是否可以检测其他样本类型？
试剂盒可以兼容检测细胞、细菌及血清（浆）等样本类型
● 为了便于高浓度样本检测，标曲线性范围是否可以扩大？
可将标曲线性范围调整至 0.15625-10 nmol/mL，取 50 μL 0.25 mM 标准品，加入 1.2 mL提取缓冲液，即得 10 nmol/mL标准品，可参考标准品配制部分进行梯度稀释。
● 检测过程中荧光信号是否稳定？会不会随时间衰减而影响定量结果？
不会显著衰减。在本试剂盒推荐的 37℃反应条件下，连续检测 3 小时内，样本和标准曲线的荧光信号波动均控制在 20%以内。这表明荧光信号在 3 小时内保持高度稳定，不会因信号衰减影响酶活定量结果。
● 肝脏样本可以手动匀浆吗？
可以。推荐使用玻璃匀浆器在冰浴中手动操作，注意保持研磨力度一致并低温保护酶活性，样本量较少时尤为适用。
The cytochrome P450 enzyme superfamily consists of heme-containing monooxygenases widely distributed in organisms, which play critical roles in drug metabolism, xenobiotic detoxification and endogenous substance biosynthesis. Its most prominent characteristics include broad substrate specificity, diverse catalytic reactions (such as hydroxylation, dealkylation, epoxidation, etc.), as well as an electron transport chain dependent on reduced nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen. Distinct P450 isoforms possess unique tissue distribution patterns and substrate selectivity, and are commonly utilized as biomarkers for evaluating drug-drug interactions and the toxic effects of environmental pollutants.
In terms of activity detection principle, cytochrome P450 enzyme exhibits 7-ethoxycoumarin O-deethylase (ECOD) activity. It catalyzes the O-deethylation of the substrate 7-ethoxycoumarin to form 7-hydroxycoumarin, a highly fluorescent metabolite. The increase in fluorescence intensity of 7-hydroxycoumarin within the reaction system can be measured by a fluorescence microplate reader or fluorescence spectrophotometer to quantitatively reflect the catalytic activity of CYP450 enzyme.
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Notes:
1.Please check the volume of each component before the experiment.
2.An extra 10% volume of each component is provided on the basis of standard specifications for standard curve preparation or preliminary tests.
Self-supplied Instruments and Reagents
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Notes
● Before formal detection, it is recommended to select 2–3 samples with expected large differences for preliminary experiments.
● For tissue samples, cell samples and other specimens, the results between samples can be normalized by measuring protein concentration.
● This kit is compatible with spectrophotometer detection; adjust the preparation volume of detection reagents proportionally in accordance with the spectrophotometer operation requirements.
● It is recommended that users establish a standard curve by themselves to improve the accuracy of test results. If a self-established standard curve is unavailable, the typical standard curve equation provided in the Result Display section can be used for calculation.
● Biochemical detection reagents are generally irritating and biologically toxic. For personal safety and health, complete biosafety protection must be maintained throughout the experiment. Wear lab coats, masks, gloves, hair caps and other protective equipment, and conduct all operations in a fume hood or biosafety cabinet.
● This product is for research use only, not for clinical diagnosis.
Experimental Procedures
Reagent Preparation
1-1 Reagent Preparation
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1-2 Standard Preparation
Take 10 μL of 0.25 mM standard solution and mix with 90 μL Extraction Buffer for dilution to obtain 25 nmol/mL standard solution, then perform serial dilution as shown in the table below. Standard detection and standard curve preparation are required for each experiment. The diluted standard solution is unstable and must be used within 4 hours.
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2 Sample Preparation
Notes:
1. Fresh samples are recommended, and extraction should be performed as soon as possible. If extraction cannot be completed on the same day, intact cells or aliquoted tissue blocks can be stored at -80 °C and used within one month. When starting the experiment, thaw samples on ice after taking them out from -80 °C. Note that this treatment may compromise sample stability and lead to lower-than-expected test results.
2. Liver tissue contains abundant cytochrome P450 with high enzyme activity. The supernatant can be diluted 20–40 times with Extraction Buffer prior to detection.
● Animal tissue: Weigh approximately 0.1 g tissue sample, add 1 mL Extraction Buffer, homogenize on ice, centrifuge at 10000 g and 4 °C for 10 min, and collect the supernatant for testing.
3 Experimental Procedures
1. Microplate reader preparation: Preheat the device for more than 30 min, and set excitation wavelength to 380 nm and emission wavelength to 450 nm.
2. Working reagent preparation: Mix Reagent Ⅲ and Extraction Buffer at a volume ratio of 1:4; prepare right before use.
3. Preparation of working solutions: Standard working solution: Prepare 100 µL for each standard/blank well, freshly prepared before use. Pipette 90 µL Reagent Ⅰ and 10 µL Reagent Ⅱ. Store the prepared solution away from light and mix thoroughly before use.
Test working solution: Prepare 100 µL for each test well, freshly prepared before use. Pipette 80 µL Reagent Ⅰ, 10 µL Reagent Ⅱ and 10 µL working reagent. Store the prepared solution away from light and mix thoroughly before use.
Control working solution: Prepare 100 µL for each control well, freshly prepared before use. Pipette 80 µL Reagent Ⅰ, 10 µL deionized water and 10 µL working reagent. Store the prepared solution away from light and mix thoroughly before use.
4. Detection system setup:
4. Detection system setup: One control well must be set for each test well. Blank and standard wells only need to be tested once. All operations should be carried out under light-shielded conditions. Perform the following operations in a black 96-well microplate:
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● Can the linear range of the standard curve be expanded for high-concentration samples?
The linear range can be adjusted to 0.15625–10 nmol/mL. Take 50 μL of 0.25 mM standard and mix with 1.2 mL Extraction Buffer to prepare a 10 nmol/mL standard solution, then perform serial dilution following the Standard Preparation section.
● Is the fluorescence signal stable during detection? Will signal decay affect quantitative results over time?
No obvious signal decay occurs. Under the recommended 37 °C incubation condition of this kit, the fluorescence signal fluctuation of samples and standard curves is controlled within 20% within 3 consecutive hours, indicating high signal stability that will not interfere with enzyme activity quantification.
● Can manual homogenization be used for liver tissue samples?
Yes. A glass homogenizer is recommended for manual homogenization on ice. Maintain consistent grinding force and low temperature to preserve enzyme activity, especially suitable for small sample amounts.
| 组分货号 | 组分名称 | 物理外观 | 48T | 96T | 储存 |
| C1526057A | 提取缓冲液 | 液体 | 70 mL | 70 mL×2 | 2-8 ℃ |
| C1526057B | 试剂一 | 液体 | 10 mL | 20 mL | 2-8 ℃ |
| C1526057C | 试剂二 | 液体 | 5 mL | 10 mL | -20℃. 避光 |
| C1526057D | 试剂三 | 粉末 | 1 瓶 | 1 瓶 | -20℃. 避光 |
| C1526057E | 试剂四 | 液体 | 10 mL | 20 mL | 2-8 ℃ |
| C1526057F | 0.25 mM 标准品 | 液体 | 500 μL | 1 mL | -20℃. 避光 |
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