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产品介绍
Malachite Green Phosphate Assay Kit，即孔雀绿磷酸盐检测试剂盒，是一种快速、便捷、高灵敏地测定溶液中可溶性自由无机磷酸根试剂盒。本试剂盒可用于溶液中磷酸盐浓度的测定。检测原理是孔雀绿(Malachite green)、钼酸盐(Molybate)和游离的无机正磷酸盐(Orthophosphate)形成绿色磷钼酸复合物(Molybdophosphoric acid complex)，而这种绿色磷钼酸复合物在620-640 nm处的吸光度与溶液中游离的无机磷酸盐浓度成正比。本试剂盒提供了一种高灵敏度定量检测无机游离磷酸盐的可靠方法，但本试剂盒只适合于直接测定溶液中的自由磷酸根，对于脂和蛋白所结合的磷酸根必须在测定之前采用一定的方法进行释放才可以检测。
使用方法
1. 磷酸盐标准品的制备。
将5mM磷酸盐标准液配制成50μM磷酸盐工作液。例如取10μl的5mM磷酸盐标准液，加入990μl标准品稀释液，即得1ml 50μM磷酸盐工作液。标准品稀释液优先推荐使用去离子水或双蒸水。稀释后的工作液可在4ºC至少稳定一天。若使用自行准备的试剂稀释标准品，需确保该试剂没有磷酸盐污染。
2. 显色剂的配制。
根据样本的数量，计算需要配制显色剂的体积。Malachite Green Reagent A和Malachite Green Reagent B的使用比例为36:17。该显色剂在4ºC避光保存，可稳定一天。
3. 标准曲线的设置。
按照下表配制0、1、2.5、5、10、20、30、40、50 μM磷酸盐标准品。
| NO | 50μM Phosphate（μl） | H₂O（μl） | Phosphate（μM） | Phosphate（nmol/200μl） |
| 1 | 0 | 200 | 0 | 0 |
| 2 | 4 | 196 | 1 | 0.2 |
| 3 | 10 | 190 | 2.5 | 0.5 |
| 4 | 20 | 180 | 5 | 1 |
| 5 | 40 | 160 | 10 | 2 |
| 6 | 80 | 120 | 20 | 4 |
| 7 | 120 | 80 | 30 | 6 |
| 8 | 160 | 40 | 40 | 8 |
| 9 | 200 | 0 | 50 | 10 |
4. 磷酸盐浓度的检测。
a. 加入适量体积的待测样品到1.5ml离心管中，若样品体积不足200μL，则用标准品稀释液补齐至200μL，记录样品此时的稀释倍数。
b. 从每管中依次吸取200μL不同浓度的磷酸盐标准品和待测样品加到96孔板的样品孔中。
c. 各孔加入50μL显色剂，使用多孔板混匀设备混匀或使用移液器轻轻吹打混匀，37℃静置30min。
d. 用酶标仪测定630nm的吸光度。
e. 根据标准曲线和待测样品的稀释倍数计算出样品的磷酸盐浓度。
注意事项：
1. 显色试剂须现配现用，配制后可以在4ºC保存一天。试剂在使用前必须恢复至室温。
2. 本实验测定波长在620-660 nm之间，630nm最佳。如果没有酶标仪，也可使用分光光度计进行测定。在这种情况下，需按比例调整显色剂、样本、标准品的体积，使最终待测体积符合比色皿的最小检测体积。
3. 本说明书中所述的加样体积以96孔板为例，若选择比色皿或384孔板，应按比例调整体积。
4. 实验室清洁剂含有高浓度的磷酸盐，需确保实验器皿彻底清洗，防止磷酸盐污染。
5. 为获得更好的检测效果，推荐对于所有的磷酸盐标准品和待测样品设置平行孔或三复孔。
6. 如果检测出来的吸光度偏低，可能是样品中磷酸盐含量较低，需要考虑在本试剂盒许可的范围内加大样品的用量。
7. 孔雀绿试剂 A呈强酸性，有腐蚀性，操作时请小心，并确保有效防护以避免直接接触人体，并须注意避免腐蚀其它物品。
8. 本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
9. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product Introduction
Malachite Green Phosphate Assay Kit is a rapid, convenient and highly sensitive kit for determining soluble free inorganic phosphate in solutions. It can be used for the quantification of phosphate concentration in various solutions.
The detection principle is based on the reaction that malachite green, molybate and free inorganic orthophosphate form a green molybdophosphoric acid complex. The absorbance of this green complex at the wavelength of 620–640 nm is directly proportional to the concentration of free inorganic phosphate in the solution.
This kit provides a reliable method for the high-sensitivity quantitative detection of free inorganic phosphate. However, it is only suitable for the direct determination of free phosphate in solutions. For phosphate bound to lipids or proteins, the phosphate must be released by appropriate methods prior to the assay.
Product Components
1.Preparation of Phosphate Standard Solution
Dilute the 5 mM phosphate standard solution to prepare a 50 μM phosphate working solution. For example, take 10 μl of the 5 mM phosphate standard solution and add 990 μl of standard diluent to obtain 1 ml of 50 μM phosphate working solution. Deionized water or double-distilled water is preferred as the standard diluent. The diluted working solution can be stable for at least one day at 4 ºC. If self-prepared reagents are used to dilute the standard solution, ensure that the reagents are free of phosphate contamination.
2.Preparation of Chromogenic Reagent
Calculate the required volume of chromogenic reagent based on the number of samples. The ratio of Malachite Green Reagent A to Malachite Green Reagent B for use is 36:17. The chromogenic reagent can be stored at 4 ºC in the dark and remains stable for one day.
Setup of Standard Curve
3.Prepare phosphate standard solutions at concentrations of 0, 1, 2.5, 5, 10, 20, 30, 40 and 50 μM according to the table below.
| NO | 50μM Phosphate（μl） | H₂O（μl） | Phosphate（μM） | Phosphate（nmol/200μl） |
| 1 | 0 | 200 | 0 | 0 |
| 2 | 4 | 196 | 1 | 0.2 |
| 3 | 10 | 190 | 2.5 | 0.5 |
| 4 | 20 | 180 | 5 | 1 |
| 5 | 40 | 160 | 10 | 2 |
| 6 | 80 | 120 | 20 | 4 |
| 7 | 120 | 80 | 30 | 6 |
| 8 | 160 | 40 | 40 | 8 |
| 9 | 200 | 0 | 50 | 10 |
4. Determination of Phosphate Concentration
a. Add an appropriate volume of the sample to be tested into a 1.5 ml centrifuge tube. If the sample volume is less than 200 μL, make up the volume to 200 μL with standard diluent and record the dilution factor of the sample at this time.
b. Pipette 200 μL of phosphate standard solutions with different concentrations and samples to be tested from each tube in sequence into the sample wells of a 96-well plate.
c. Add 50 μL of chromogenic reagent to each well, mix thoroughly using a multi-well plate shaker or gently by pipetting up and down, and incubate at 37 ºC for 30 minutes.
d. Measure the absorbance at 630 nm using a microplate reader.
e. Calculate the phosphate concentration of the sample based on the standard curve and the dilution factor of the sample to be tested.
Notes
1.The chromogenic reagent must be prepared and used immediately. It can be stored at 4 ºC for one day after preparation. The reagent must be equilibrated to room temperature before use.
2.The detection wavelength of this experiment ranges from 620 to 660 nm, with 630 nm being optimal. If a microplate reader is not available, a spectrophotometer can be used instead. In this case, adjust the volumes of the chromogenic reagent, samples and standard solutions proportionally to ensure the final volume to be tested meets the minimum detection volume of the cuvette.
3.The sample volumes described in this instruction manual are based on a 96-well plate. If cuvettes or 384-well plates are used, the volumes should be adjusted proportionally.
4.Laboratory detergents contain high concentrations of phosphate. Ensure that the experimental utensils are thoroughly cleaned to prevent phosphate contamination.
5.For better detection results, it is recommended to set up duplicate or triplicate wells for all phosphate standard solutions and samples to be tested.
6.If the measured absorbance is low, it may be due to the low phosphate content in the sample. Consider increasing the sample dosage within the allowable range of this kit.
7.Malachite Green Reagent A is strongly acidic and corrosive. Handle it with care during operation, ensure effective protection to avoid direct contact with the human body, and take care to prevent corrosion of other items.
8.This product is intended for scientific research use by professionals only. It is not for clinical diagnosis or treatment, nor for use in food or pharmaceuticals, and should not be stored in ordinary residential premises.
9.For your safety and health, wear a lab coat and disposable gloves during operation.
| P1506503 | Components | 500T | 1000T | 2500T | Storage |
| P1506503A | 孔雀绿试剂 A | 20 mL | 40 mL | 100 mL | 2-8℃ |
| P1506503B | 孔雀绿试剂 B | 10 mL | 20 mL | 50 mL | 2-8℃ |
| P1506503C | 5mM 标准品 | 0.5 mL | 1 mL | 2.5 mL | 2-8℃ |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939531 |
分析证书 | P1506503 |
| ZJ26F0939530 |
分析证书 | P1506503 |
| ZJ26F0939529 |
分析证书 | P1506503 |
| ZJ26F0433901 |
分析证书 | P1506503 |
危险品化学品经营许可证（带存储）