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,室温,-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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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。
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 120T | 储存 | 单次测试使用量 |
| A1518297WT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法）(2-8℃组分) | A1518297A | 抗酒石酸酸性磷酸酶检测缓冲液 | 无色澄清透明溶液，无沉淀物 | 30 mL | 2-8℃. | 按需 |
| A1518297C | 酒石酸溶液 | 无色澄清透明溶液，无沉淀物 | 1 mL | 2-8℃. | 4 µL | ||
| A1518297DT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法） (-20℃组分) | A1518297B | 对硝基苯磷酸二钠 | 淡黄色粉末 | 1 EA×2 | -20℃, 避光. | 40 µL |
| A1518297D | 对硝基苯酚 (10 mM) | 无色/淡黄色透明溶液，无沉淀物 | 0.5 mL | -20℃, 避光. | 20 µL | ||
| A1518297NT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法） (常温组分) | A1518297E | 终止液 | 无色澄清透明溶液，无沉淀物 | 20 mL | Room temperature. | 160 µL |
3. 配制系列标准品
取出对硝基苯酚 (10 mM) 恢复至室温，按 对硝基苯酚 (10 mM)： 抗酒石酸酸性磷酸酶检测缓冲液 =1: 19的比例混合，使浓度达到 0.5 mM，按下表继续稀释：
加入物（μL） | 1 | 2 | 3 | 4 | 5 | 6 |
对硝基苯酚 (0.5 mM) | 2 | 4 | 8 | 12 | 16 | 20 |
抗酒石酸酸性磷酸酶检测缓冲液 | 18 | 16 | 12 | 8 | 4 | 0 |
标准品浓度 (mM) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
加入物（μL） | 空白孔 | 标准孔 | 测定孔 | 对照孔 |
抗酒石酸酸性磷酸酶检测缓冲液 | 20 | — | — | 40 |
系列标准品 (1-6号) | — | 20 | — | — |
待测样品 | — | — | 20 | 20 |
显色工作液 | 40 | 40 | 40 | — |
酒石酸溶液 | 4 | 4 | 4 | 4 |
5. TRAP测定
轻轻混匀，37 ℃孵育10 分钟，每孔加入160 μL终止液终止反应；酶标仪测定 405 nm 处各管的吸光度。
注意事项
1. 待测样品中不能含有磷酸酶抑制剂，同时需避免反复冻融。
2. 如果待测样品体积较少，可减少样品加样量，以抗酒石酸酸性磷酸酶检测缓冲液补至20 μL。
3. 如果无法检测 405 nm，亦可检测 400-415 nm 范围内吸光度。
4. 建议每次测定时都做标准曲线，以使标准更准确，另外标准品避免反复冻融。
5. 待测样品中抗酒石酸酸性磷酸酶活性较低时，可适当延长孵育时间至 30 分钟。
6. 检测组织和细胞样品时，需测定总蛋白浓度，推荐使用BCA蛋白定量试剂盒。
7. 配制1支显色工作液后应当日用完，因此请注意适当多准备一些样品一起检测。
8. 对硝基苯酚溶液对人体有害， 对硝基苯酚有腐蚀性，请小心操作。
9. 如果希望进行酶活性的绝对定量，进行酶反应时应精确计时，此时推荐采用孵育30 min或更长时间，以减小操作过程中的时间误差。
10. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Acid phosphatase (ACP) is a widely distributed enzyme found in various tissues throughout the body. It is primarily localized in cellular lysosomes and is therefore commonly used as a lysosomal marker enzyme. Additionally, ACP is present in the endoplasmic reticulum and cytoplasm. The optimal pH range for this enzyme is 4.5 to 5.5, and its characteristics vary among different animal species.
Tartrate-resistant acid phosphatase (TRAP) is a specific isoform of ACP. It is found within cytoplasmic vacuoles in normal human alveolar macrophages and in the spleen of patients with leukemia, and is not typically released into the bloodstream. TRAP in the blood originates mainly from osteoclasts. Therefore, measuring blood TRAP levels can reflect osteoclast activity and is widely regarded as the only hematological indicator for assessing bone resorption activity in the body. TRAP is a glycosylated, metal-containing enzyme highly expressed in osteoclasts and chondroclasts, and is also expressed in activated macrophages and neurons.
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 120T | Storage | Quantity Per Test |
| A1518297WT | Tartrate Resistant Acid Phosphatase (TRAP) Activity Assay Kit (pNPP, Micro Method) (2-8℃ Components) | A1518297A | TRAP Assay Buffer | Colorless transparent solution, free of sediment | 30 mL | 2-8℃ | As needed |
| A1518297C | Tartrate Solution | Colorless transparent solution, free of sediment | 1 mL | 2-8℃ | 4 µL | ||
| A1518297DT | Tartrate Resistant Acid Phosphatase (TRAP) Activity Assay Kit (pNPP, Micro Method)(-20℃ Components) | A1518297B | pNPP | Light yellow powder | 1 EA×2 | 20℃. Store in the dark. | 40 µL |
| A1518297D | p-nitrophenol (10mM) | Colorless/light yellow transparent solution, free of sediment | 0.5 mL | 20℃. Store in the dark. | 20 µL | ||
| A1518297NT | Tartrate Resistant Acid Phosphatase (TRAP) Activity Assay Kit (pNPP, Micro Method)(RT Components) | A1518297E | Stopping Solution | Colorless transparent solution, free of sediment | 20 mL | Room temperature | 160 µL |
Assay Principle
Under acidic conditions, ACP acts on p-nitrophenyl phosphate (pNPP), hydrolyzing it to form p-nitrophenol (p-NP). In an alkaline environment, p-NP produces a yellow color, with the intensity being directly proportional to the enzyme activity, showing maximum absorbance at 405 nm. By adding an appropriate amount of tartrate to the reaction system, the activity of other ACP isoforms is inhibited, and the measured ACP activity specifically represents TRAP activity. TRAP activity levels are calculated by measuring the amount of p-NP generated.
Applicable Samples
Cell or tissue lysates, homogenates, plasma, serum, urine, etc.
Reagents, consumables and Equipments not provided
Microplate reader (capable of measuring absorbance at 405 nm)
96-well plate, adjustable pipettes and tips, centrifuge tubes
Centrifuge, water bath or incubator
PBS or normal saline
Assay Procedure (For Reference Only)
1. Sample Preparation
1.1 Plasma, Serum, and Urine Samples
Plasma and serum prepared by routine methods can be used directly for the assay. Urine can also typically be used directly. If not assayed immediately, samples can be stored at -80°C but avoid repeated freeze-thaw cycles.
1.2 Tissue Samples
Homogenize tissue samples using a ratio of tissue weight (g) : normal saline volume (mL) = 1:10. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.
1.3 Cell Samples
Homogenize cell samples using a ratio of cell number (10⁶) : normal saline volume (mL) = 5:1. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.
Note: Before formal testing, perform a preliminary experiment using 2-3 samples with expected activity differences, diluted to various concentrations. Based on the preliminary results and the kit's linear range (5.0-300 U/L), dilute samples appropriately with PBS or normal saline before the final assay.
2. Preparation of Working Color Solution
Briefly centrifuge one vial of pNPP. Carefully open the vial, add 0.25 mL of deionized water, close the cap tightly, and dissolve completely by vortexing. Keep on ice until use. Any unused portion can be stored at -20°C protected from light for up to 2 days. To prepare the Working Color Solution, dilute the dissolved pNPP with TRAP Assay Buffer at a ratio of 1:20. Mix well, keep on ice, and protect from light. The freshly prepared Working Color Solution is stable for 6 hours.
3. Preparation of Standard Curve Dilutions
Allow the p-nitrophenol (10 mM) to equilibrate to room temperature. Dilute it with TRAP Assay Buffer at a ratio of 1:19 to achieve a concentration of 0.5 mM. Then, prepare serial dilutions according to the table below:
| Addition (μL) | 1 | 2 | 3 | 4 | 5 | 6 |
|---|---|---|---|---|---|---|
| p-nitrophenol (0.5 mM) | 2 | 4 | 8 | 12 | 16 | 20 |
| TRAP Assay Buffer | 18 | 16 | 12 | 8 | 4 | 0 |
| Standard Concentration (mM) | 0.05 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
4. TRAP Assay Setup
Set up Blank, Standard, Test, and Control wells in a 96-well plate as described below. Add reagents in the order listed, avoiding bubbles. If sample TRAP activity is expected to be very high, reduce the sample volume or dilute the sample appropriately. It is recommended to run samples in duplicate.
| Addition (μL) | Blank Well | Standard Wells | Test Well | Control Well |
|---|---|---|---|---|
| TRAP Assay Buffer | 20 | — | — | 40 |
| Serial Standards (1~6) | — | 20 | — | — |
| Test Sample | — | — | 20 | 20 |
| Working Color Solution | 40 | 40 | 40 | — |
| Tartrate Solution | 4 | 4 | 4 | 4 |
Note: The Control well corrects for background absorbance from the sample itself and non-specific reactions. For final calculations, subtract the Control well absorbance from the Test well absorbance (ΔA405).
5. Measurement
Mix gently. Incubate at 37°C for 10 minutes. Stop the reaction by adding 160 μL of Stopping Solution to each well. Measure the absorbance of each well at 405 nm using a microplate reader.
6. Calculation of Results
Plot the standard curve using the concentrations of the serial standards (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM) as the x-axis and their corresponding absorbance values (Standard well OD - Blank well OD) as the y-axis. Perform linear regression to obtain the equation y = ax + b.
Determine the amount of p-nitrophenol generated in the Test sample, x (in mM), from the standard curve using the formula x = (ΔA405- b) / a.
Definition of Enzyme Activity Unit
For Tissue and Cell Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per gram of protein at pH 4.8 and 37°C.
Formula: TRAP Activity (U/g prot) = x×1000×N÷t÷Cpr
For Serum (Plasma) and Urine Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per liter of serum (plasma) or urine at pH 4.8 and 37°C.
Formula: TRAP Activity (U/L) = x×1000×N÷t
Parameter Description:
a: Slope of the standard curve
b: Intercept of the standard curve
N: Dilution factor of the sample before addition to the reaction
1000: Unit conversion factor (1 mmol/L = 1000 μmol/L)
Cpr: Protein concentration of the sample (g prot/L)
t: Enzymatic reaction time (min)
ΔA405: Absolute OD of the sample (Test OD - Control OD)
y: Standard OD - Blank OD
Notes
Test samples must not contain phosphatase inhibitors. Avoid repeated freeze-thaw cycles.
If the sample volume is limited, reduce the sample addition volume and adjust the total volume to 20 μL with TRAP Assay Buffer.
If measurement at 405 nm is not possible, absorbance in the range of 400-415 nm is also acceptable.
It is recommended to generate a standard curve for each assay for optimal accuracy. Avoid repeated freeze-thaw cycles of the standard.
If TRAP activity in the sample is expected to be low, the incubation time can be extended up to 30 minutes.
When assaying tissue or cell samples, total protein concentration must be determined. A BCA protein assay kit is recommended.
Prepare only enough Working Color Solution for same-day use. It is advisable to plan experiments to assay multiple samples together.
p-nitrophenol solution is harmful. Stopping Solution is corrosive. Please handle with care. Wear laboratory coat and disposable gloves for safety and health protection.
For absolute quantification of enzyme activity, precise timing of the enzymatic reaction is critical. Incubating for 30 minutes or longer is recommended to minimize timing errors.
This product is for research use only and is not intended for diagnostic or therapeutic applications.
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 120T | Storage | 单次测试使用量 |
| A1518297WT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法）(2-8℃组分) | A1518297A | 抗酒石酸酸性磷酸酶检测缓冲液 | 无色澄清透明溶液，无沉淀物 | 30 mL | 2-8℃. | 按需 |
| A1518297C | 酒石酸溶液 | 无色澄清透明溶液，无沉淀物 | 1 mL | 2-8℃. | 4 µL | ||
| A1518297DT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法） (-20℃组分) | A1518297B | 对硝基苯磷酸二钠 | 淡黄色粉末 | 2×1 EA | -20℃, 避光. | 40 µL |
| A1518297D | 对硝基苯酚 (10 mM) | 无色/淡黄色透明溶液，无沉淀物 | 0.5 mL | -20℃, 避光. | 20 µL | ||
| A1518297NT | 抗酒石酸酸性磷酸酶 (TRAP) 活性检测试剂盒（pNPP, 微量法） (常温组分) | A1518297E | 终止液 | 无色澄清透明溶液，无沉淀物 | 20 mL | 室温 | 160 µL |
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 |
|---|---|---|
 ZJ26F0939071 |
分析证书 | A1518297 |
| ZJ26F0939072 |
分析证书 | A1518297 |
| ZJ26F0939073 |
分析证书 | A1518297 |
| ZJ26F0434311 |
分析证书 | A1518297 |
危险品化学品经营许可证（带存储）