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。
蛋白质羰基是多种氨基酸在蛋白质氧化修饰过程中的早期标志，其含量高低表明蛋白质氧化损伤程度的大小，是衡量蛋白质氧化损伤的主要指标。
检测原理：羰基与2,4-二硝基苯肼反应生成红色2,4-二硝基苯腙，在370nm处有特征吸收峰。
适用样本： 动植物组织、细胞、血清、血浆、细菌
自备仪器和试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测370 nm处的吸光度 |
| 耗材 | 96孔酶标板 | UV板 |
| 试剂 | PBS/无水乙醇、乙酸乙酯 | 清洗样本/检测用 |
| 其他 | 匀浆器（组织样本）、恒温箱、冰盒、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B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
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。
操作步骤（仅供参考）
1. 试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| 提取缓冲液 | 即用型；使用前平衡到室温 | 4℃保存 |
| 抗氧化剂工作液 | 临用前配制 称取0.1 g加1 mL去离子水溶解配制，每1 mL为10个样品用量 | 现用现配 有一定毒性，注意防护 |
| 色原 | 即用型；使用前平衡到室温 | 4℃避光保存 有一定毒性，注意防护 |
| 试剂D | 即用型；使用前平衡到室温 | 4℃保存 |
| 三氯乙酸 | 即用型；使用前平衡到室温 | 4℃保存 有刺激性气味，建议在通风橱进行实验 |
| 盐酸胍 | 即用型；使用前平衡到室温 | 4℃保存 有刺激性气味，建议在通风橱进行实验 |
2. 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。
2.1 动植物组织
称取约0.1 g样本，加入1 mL 提取缓冲液，冰浴匀浆，4000g，4℃离心10 min，取上清液，加0.1 mL 抗氧化剂工作液，室温放置10 min后，10000 g，4℃离心10 min，取上清液， 置冰上待测。
2.2 细胞或细菌样本
收集5×10⁶个细胞，用冷的PBS清洗细胞或细菌，800 g离心2 min后弃上清，加入1 mL 提取缓冲液，冰浴超声波破碎5 min（功率20%或200 W，超声3 s，间隔7 s，重复30次），然后10000 g，4℃离心10 min，取上清液，置冰上待测。
2.3 血清（浆）
直接测定。
3. 实验步骤
3.1 酶标仪准备
预热30 min以上，调节波长到370 nm。
3.2 检测体系配制
在1.5 mL EP管中按照如下方式操作，每个测定管均需设置一个对照管。
| 试剂 | 对照管（μL） | 测定管（μL） |
| 样本 | 60 | 60 |
| 色原 | 0 | 120 |
| 试剂D | 120 | 0 |
| 三氯乙酸 | 150 | 150 |
| 无水乙醇 | 150 | 150 |
| 乙酸乙酯 | 150 | 150 |
| 盐酸胍 | 300 | 300 |
3.3 吸光度检测
取上清200 μL于96孔UV板中，370 nm处测定吸光值，分别标记为A对照和A测定。
4. 结果计算
4.1 数据处理
计算ΔA=A测定-A对照 。
4.2 样本蛋白质羰基含量计算
按样本质量计算
蛋白质羰基含量 (μmol/g)=[ΔA×V反总÷(ε×d)]÷(W×V样÷V样总)×n=0.454×ΔA÷W×n
按细胞或细菌数量计算
蛋白质羰基含量 (μmol/10⁴)=[ΔA×V反总÷(ε×d)]÷(N×V样÷V样总)×n=0.454×ΔA÷N×n
按液体体积计算
蛋白质羰基含量 (μmol/mL)=[ΔA×V反总÷(ε×d)]÷V样×n=0.454×ΔA×n
按蛋白浓度数量计算
蛋白质羰基含量 (μmol/mg prot)=[ΔA×V反总÷(ε×d)]÷(V样×Cpr)×n=0.454×ΔA÷Cpr×n
参数说明：
V反总：反应体系总体积，0.3mL；
ε：羰基摩尔消光系数，22 mL/μmol/cm；
d：96孔板光径，0.5 cm；
V样：加入样本体积，0.06 mL；
V样总：加入提取缓冲液体积，1 mL；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
N：细胞或细菌数量，以10⁴为单位（例如细胞数量为5×10⁶，N=500）；
n：样本稀释倍数。
结果展示
取 0.1 g 小鼠肾脏，按照测定步骤操作，用 96 孔 UV 板进行检测。测得ΔA=A测定-A对照=0.103-0.071=0.032。按样本质量计算得：蛋白质羰基含量(μmol/g)=[ΔA×V反总÷(ε×d)]÷(W×V样÷V样总)×n=0.454×ΔA÷W×n=0.145 μmol/g。
常见问题及解答
Q：检测得到的样本ΔA过高或者过低怎么办？
A：如果样本ΔA>0.8，则说明样本蛋白质羰基含量太高，需用提取缓冲液适当稀释（计算公式中乘以相应稀释倍数）或减少提取用样本量。如果样本的ΔA＜0.005，可适当提高样本量。
Protein carbonyls are early markers of protein oxidation modification involving various amino acids. Their level directly indicates the extent of protein oxidative damage and serves as a primary index for assessing protein oxidative injury.
Detection Principle
Carbonyl groups react with 2,4-dinitrophenylhydrazine (DNPH) to form red-colored 2,4-dinitrophenylhydrazone, which exhibits a characteristic absorption peak at 370 nm.
Applicable Samples
Animal/plant tissues, cells, serum, plasma, bacteria.
Reagents, consumables and Equipments not provided
Resource Type | Resource Name | Remarks |
|---|---|---|
Instrument | Microplate Reader | Capable of measuring absorbance at 370 nm |
Consumable | 96-Well Microplate | UV-transparent plate |
Reagent | PBS / Anhydrous Ethanol, Ethyl Acetate | For sample washing / assay use |
Others | Homogenizer (for tissue), Incubator, Ice Box, Refrigerated Centrifuge, Adjustable Pipettes and Tips | Using a multichannel pipette can improve efficiency for large sample batches |
Precautions
Prior to formal testing, it is recommended to perform a preliminary experiment using 2-3 samples with expected significant differences.
For tissue and cell samples, normalizing results between samples by measuring protein concentration is recommended. Aladdin B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit are recommended.
This kit is compatible with spectrophotometer detection. Please adjust the reagent preparation volumes proportionally according to spectrophotometer cuvette requirements.
Biochemical assay reagents generally possess irritant properties or biological toxicity. For your safety and health, please ensure proper biosafety protection throughout the experiment by wearing lab coats, masks, gloves, and head covers, and conduct experiments in a fume hood or biosafety cabinet.
This product is for scientific research use only and is not intended for clinical diagnosis.
Protocol (For Reference Only)
1. Reagent Preparation
Reagent Name | Preparation Method | Notes |
|---|---|---|
Extraction Buffer | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C |
Working Antioxidant | Prepare immediately before use. Dissolve 0.1 g in 1 mL deionized water. Each 1 mL is sufficient for 10 samples. | Prepare fresh. Toxic, use with caution and proper protection. |
Chromogen | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. Toxic, use with caution and proper protection. |
Reagent D | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C |
TCA | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C. Irritating odor; recommended to use in a fume hood. |
Guanidine Hydrochloride | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C. Irritating odor; recommended to use in a fume hood. |
2. Sample Preparation
*Note: Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.*
2.1 Animal/Plant Tissues
Weigh approximately 0.1 g of tissue sample. Add 1 mL of Extraction Buffer. Homogenize on ice. Centrifuge at 4000 × g for 10 min at 4°C. Collect the supernatant. Add 0.1 mL of Working Antioxidant. Incubate at room temperature for 10 min. Centrifuge at 10000 × g for 10 min at 4°C. Collect the supernatant and keep on ice for testing.
2.2 Cell or Bacterial Samples
Collect 5 × 10⁶ cells. Wash the cells or bacteria with cold PBS. Centrifuge at 800 × g for 2 min and discard the supernatant. Add 1 mL of Extraction Buffer. Sonicate on ice for 5 min (20% power or 200 W, 3 s on, 7 s off, repeat 30 times). Centrifuge at 10000 × g for 10 min at 4°C. Collect the supernatant and keep on ice for testing.
2.3 Serum (Plasma)
Test directly.
3. Assay Procedure
3.1 Microplate Reader Setup
Preheat the microplate reader for at least 30 min. Set the wavelength to 370 nm.
3.2 Reaction System Setup
Prepare the reaction in 1.5 mL microcentrifuge tubes as described below. Each test sample requires a corresponding control tube.
Reagent | Control Tube (μL) | Test Tube (μL) |
|---|---|---|
Sample | 60 | 60 |
Chromogen | 0 | 120 |
Reagent D | 120 | 0 |
TCA | 150 | 150 |
Anhydrous Ethanol | 150 | 150 |
Ethyl Acetate | 150 | 150 |
Guanidine Hydrochloride | 300 | 300 |
3.3 Absorbance Measurement
Transfer 200 μL of the supernatant to a UV-transparent 96-well plate. Measure the absorbance at 370 nm. Record the values as AControl and ATest.
4. Calculation of Results
4.1 Data Processing
Calculate ΔA = ATest - AControl.
4.2 Calculation of Sample Protein Carbonyl Content
Based on Sample Mass
Protein Carbonyl Content (μmol/g) = [ΔA × VRTotal ÷ (ε × d)] ÷ (W × VS ÷ VSTotal) × n = 0.454 × ΔA ÷ W × n
Based on Cell or Bacterial Count
Protein Carbonyl Content (μmol/10⁴ cells) = [ΔA × VRTotal ÷ (ε × d)] ÷ (N × VS ÷ VSTotal) × n = 0.454 × ΔA ÷ N × n
Based on Liquid Volume
Protein Carbonyl Content (μmol/mL) = [ΔA × VRTotal ÷ (ε × d)] ÷ VS × n = 0.454 × ΔA × n
Based on Protein Concentration
Protein Carbonyl Content (μmol/mg prot) = [ΔA × VRTotal ÷ (ε × d)] ÷ (VS × Cpr) × n = 0.454 × ΔA ÷ Cpr × n
Parameter Definitions:
VRTotal: Total volume of the reaction system (0.3 mL)
ε: Molar extinction coefficient of carbonyl (22 mL/μmol/cm)
d: Light path of the 96-well plate (0.5 cm)
VS: Volume of sample added (0.06 mL)
VSTotal: Volume of Extraction Buffer added for homogenization (1 mL)
Cpr: Protein concentration of the sample (mg/mL)
W: Sample mass (g)
N: Cell or bacterial count (unit: 10⁴ cells. Example: for 5 × 10⁶ cells, N = 500)
n: Sample dilution factor.
Example of Results
Mouse kidney (0.1 g) was processed according to the protocol and measured using a UV-transparent 96-well plate. The measured values were ΔA = ATest - AControl = 0.103 - 0.071 = 0.032.
Calculation based on sample mass:
Protein Carbonyl Content (μmol/g) = [ΔA × VRTotal ÷ (ε × d)] ÷ (W × VS ÷ VSTotal) × n = 0.454 × ΔA ÷ W × n = 0.145 μmol/g.
Frequently Asked Questions (FAQ)
Q: What should I do if the measured ΔA of the sample is too high or too low?
A: If ΔA > 0.8, it indicates the protein carbonyl content in the sample is too high. The sample should be appropriately diluted with Extraction Buffer (multiply by the corresponding dilution factor in the calculation formula) or the amount of starting sample material should be reduced. If ΔA < 0.005, the amount of starting sample material can be appropriately increased.
| P1515832 | 组件 | 物理外观 | 48T/48S | 96T/96S | Storage |
| P1515832A | 提取缓冲液 | 液体 | 60 mL | 120 mL | 2-8℃. |
| P1515832B | 抗氧化剂 | 粉末 | 1 EA (48T) | 1 EA (96T) | 2-8℃. Store in the dark. |
| P1515832C | 色原 | 液体 | 7.5 mL | 15 mL | 2-8℃. Store in the dark. |
| P1515832D | 试剂D | 液体 | 7.5 mL | 15 mL | 2-8℃. |
| P1515832E | 三氯乙酸 | 液体 | 18 mL | 36 mL | 2-8℃. |
| P1515832F | 盐酸胍 | 液体 | 36 mL | 72 mL | 2-8℃. |
请在实验前检查各组分的量。
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。
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 |
|---|---|---|
 ZJ26F0535489 |
分析证书 | P1515832 |
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