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,-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。
甘油是动植物组织以及血液中甘油三酯代谢的中间产物，甘油三酯经过水解产生甘油，甘油再进一步氧化为细胞代谢提供能量，因此，甘油含量是甘油三酯水解反应的可靠检测指标，检测更加方便。
检测原理：甘油在ATP存在下被甘油激酶磷酸化为3-磷酸甘油，再被甘油磷酸氧化酶氧化产生过氧化氢；在过氧化氢酶作用下生色底物转化为苯醌亚胺，在505nm处有吸收峰，通过测定该波长吸光度即可计算甘油的含量。
检测范围：31.25-2000 nmol/mL
灵敏度：31.25 nmol/mL
适用样本：动植物组织、细胞、血清（浆）或其他液体
注意：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。
自备仪器和试剂
酶标仪或可见光分光光度计（能测505nm处的吸光度）
96孔板或微量玻璃比色皿、可调节式移液枪及枪头、1.5mL离心管
恒温箱、制冰机、低温离心机
去离子水、PBS、异丙醇
匀浆器或研钵（如果是组织样本）
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Working Reagent Ⅰ | 临用前配制； 48T加入7mL Reagent Ⅲ，96T加入14mL Reagent Ⅲ，充分溶解待用。 | 用不完的试剂-20℃避光分装保存1个月，避免反复冻融。 |
| Working Reagent Ⅱ | 临用前配制； 48T加入7mL Reagent Ⅲ，96T加入14mL Reagent Ⅲ，充分溶解待用。 | 4℃避光保存，可稳定1个月。 |
| Working Reagent | 临用前配制； 将Working Reagent Ⅰ和Working Reagent Ⅱ按照1: 1的比例混合均匀，现配现用。 | |
| Reagent Ⅲ | 即用型，使用前平衡到室温 | 4℃保存 |
| Standard | 即用型，使用前平衡到室温； 2 µmol/mL甘油标准品 | 4℃避光保存 |
2、标准曲线设置
使用2 µmol/mL甘油标准品，按照下表所示， 进一步稀释成标准品：
| 序号 | Standard 体积（μL） | 去离子水体积（μL） | 浓度（nmol/mL） |
| Std.1 | 200μL of 2 µmol/mL Standard | 0 | 2000 |
| Std.2 | 100μL of Std.1 | 100 | 1000 |
| Std.3 | 100μL of Std.2 | 100 | 500 |
| Std.4 | 100μL of Std.3 | 100 | 250 |
| Std.5 | 100μL of Std.4 | 100 | 125 |
| Std.6 | 100μL of Std.5 | 100 | 62.5 |
| Std.7 | 100μL of Std.6 | 100 | 31.25 |
| Blank | 0 | 100 | 0 |
注意：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。
3、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。
3.1 组织
称取约0.1g样本，用PBS冲洗组织，滤纸吸干，加入1mL异丙醇，用匀浆器或研钵冰浴匀浆，10,000g，4℃离心10min，取上清液待测。
3.2 细胞
收集500万细胞到离心管内，用冷PBS清洗细胞，离心后弃上清，加入1mL异丙醇，冰浴超声波破碎细胞5min（功率20%或200W，超声3s，间隔7s，重复30次），10,000g，4℃离心10min，取上清液待测。
3.3 血清（浆）等液体样本
直接检测。
注意：如需测定蛋白浓度，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。因异丙醇会导致蛋白变性，样本需另行用去离子水按照步骤提取。
4、实验步骤
4.1 酶标仪或可见光分光光度计预热30min以上，调节波长到505nm，可见光分光光度计用去离子水调零。
4.2 操作表（下述操作在96孔板或微量玻璃比色皿中操作）：
| 试剂 | 空白孔 (μL) | 标准孔 | 对照孔 | 测定孔 |
| 样本 | 0 | 0 | 0 | 20 |
| 异丙醇 | 0 | 0 | 20 | 0 |
| Standard | 0 | 20 | 0 | 0 |
| 去离子水 | 20 | 0 | 0 | 0 |
| Working Reagent | 200 | 200 | 200 | 200 |
4.3 立即混匀，记录505nm处吸光值A1，37°C孵育10min。迅速取出记录10min时的吸光度A2，计算ΔA=A2-A1。空白孔记为A，标准孔记为A标准，测定孔记为A测定。计算ΔΔA测定 =ΔA测定 -ΔA对照 ，ΔΔA标准 =ΔA标准 -ΔA空白 。
注意：对照孔、空白管和标准管只需做1-2次。
实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔΔA测定 小于0.004，可适当加大样本量。如果ΔΔA测定 大于1000 nmol/mL的ΔΔA标准 ，样本可用异丙醇进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
5、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
5.1 标准曲线的绘制
以标准溶液浓度为x轴，ΔΔA标准 为y轴，绘制标准曲线，得到标准方程，将ΔΔA测定 带入方程得到x (nmol/mL)。
5.2 甘油含量的计算
(1) 按蛋白浓度计算
甘油 (nmol/mg蛋白)=(V样 ×x)÷(V样 ×Cpr)=x÷Cpr
(2) 按样本鲜重计算
甘油 (nmol/g 鲜重)=(V样 ×x)÷(w×V样 ÷V样总 )=x÷w
(3) 按细胞数量计算
甘油 (nmol/10⁶ cell)=(V样 ×x)÷(n×V样 ÷V样总 )=x÷n
(4) 按液体体积计算
甘油 (nmol/mL)=(V样 ×x)÷V样 =x
参数说明
V样 ：加入反应体系中样本体积，0.005mL；
V样总 ：加入异丙醇体积，1mL；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
n：细胞数量，以10⁶计。
6、结果展示
以下数据仅供参考，实验者需根据自己的实验对样品进行检测。
甘油标准曲线：y=0.0003x-0.0018，R²=1
图1：甘油标准曲线
图2：本产品测定兔肝脏和玉米种子中甘油的含量
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、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Glycerol is an intermediate product in the metabolism of triglycerides in animal and plant tissues as well as in blood. Triglycerides are hydrolyzed to produce glycerol, which is then further oxidized to provide energy for cellular metabolism. Therefore, the glycerol content serves as a reliable indicator for monitoring triglyceride hydrolysis, offering a more convenient detection method.
Detection Principle: Glycerol is phosphorylated to glycerol-3-phosphate by glycerol kinase in the presence of ATP. Glycerol-3-phosphate is then oxidized by glycerol phosphate oxidase, generating hydrogen peroxide. Under the action of peroxidase, a chromogenic substrate is converted to a quinoneimine dye, which has an absorption peak at 505 nm. The glycerol content is calculated by measuring the absorbance at this wavelength.
Detection Range: 31.25 - 2000 nmol/mL
Sensitivity: 31.25 nmol/mL
Applicable Samples: Animal/plant tissues, cells, serum (plasma), or other liquids
Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
User-Prepared Instruments and Reagents
1.Microplate reader or visible spectrophotometer (capable of measuring absorbance at 505 nm)
2.96-well plate or micro glass cuvettes, adjustable micropipettes and tips, 1.5 mL centrifuge tubes
3.Incubator, ice maker, refrigerated centrifuge
4.Deionized water, PBS, Isopropanol
5.Homogenizer or mortar (for tissue samples)
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Notes |
| Working Reagent Ⅰ | Prepare before use: For 48T, add 7 mL Reagent III; for 96T, add 14 mL Reagent III. Dissolve completely. | Unused reagent can be aliquoted and stored at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles. |
| Working Reagent Ⅱ | Prepare before use: For 48T, add 7 mL Reagent III; for 96T, add 14 mL Reagent III. Dissolve completely. | Can be stored at 4°C protected from light for one month. |
| Working Reagent | Prepare before use: Mix Working Reagent I and Working Reagent II in a 1:1 ratio. Prepare freshly for each use. | |
| Reagent Ⅲ | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Standard | Ready-to-use; equilibrate to room temperature before use. (2 μmol/mL Glycerol Standard) | Store at 4°C protected from light. |
2. Standard Curve Setup
Using the 2 μmol/mL Glycerol Standard, prepare a dilution series as shown below:
| Tube | Standard Volume | Deionized Water Volume (μL) | Concentration (nmol/mL) |
| Std.1 | 200μL of 2 µmol/mL Standard | 0 | 2000 |
| Std.2 | 100μL of Std.1 | 100 | 1000 |
| Std.3 | 100μL of Std.2 | 100 | 500 |
| Std.4 | 100μL of Std.3 | 100 | 250 |
| Std.5 | 100μL of Std.4 | 100 | 125 |
| Std.6 | 100μL of Std.5 | 100 | 62.5 |
| Std.7 | 100μL of Std.6 | 100 | 31.25 |
| Blank | 0 | 100 | 0 |
Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month.
3.1 Tissue
Weigh approximately 0.1 g of sample. Rinse the tissue with PBS, blot dry with filter paper. Add 1 mL of isopropanol and homogenize in an ice bath using a homogenizer or mortar. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant for assay.
3.2 Cells
Collect 5 million cells into a centrifuge tube. Wash the cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of isopropanol. Disrupt the cells by sonication in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant for assay.
3.3 Serum (Plasma) and Other Liquid Samples
Assay directly.
Note: If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended. Since isopropanol denatures proteins, prepare a separate sample aliquot using deionized water according to the extraction steps for protein assay.
4. Assay Steps
4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 505 nm. For spectrophotometers, zero the instrument with deionized water.
4.2 Assay Procedure (perform in a 96-well plate or micro glass cuvette):
| Reagent | Blank Well (μL) | Standard Well (μL) | Control Well (μL) | Test Well (μL) |
| Sample | 0 | 0 | 0 | 20 |
| Isopropanol | 0 | 0 | 20 | 0 |
| Standard | 0 | 20 | 0 | 0 |
| Deionized Water | 20 | 0 | 0 | 0 |
| Working Reagent | 200 | 200 | 200 | 200 |
4.3 Mix immediately and record the initial absorbance at 505 nm (A₁). Incubate at 37°C for 10 minutes. Quickly remove and record the absorbance at 10 minutes (A₂). Calculate ΔA = A₂ - A₁ for each well. Record ΔA for the Blank well as ΔA blank , for the Standard well as ΔA standard , for the Control well as ΔA control , and for the Test well as ΔA test . Calculate ΔΔA test = ΔA test - ΔA control and ΔΔA standard = ΔA standard - ΔA blank .
Note: The Control, Blank, and Standard wells only need to be set up 1-2 times. A preliminary test with 2-3 samples showing expected significant differences is recommended. If ΔΔA test is less than 0.004, consider increasing the sample volume. If ΔΔA test is greater than the ΔΔA standard of the 1000 nmol/mL standard, further dilute the sample with isopropanol (multiply the result by the dilution factor) or reduce the sample amount used for extraction.
5. Calculation of Results
Note: We provide both derived and simplified calculation formulas. They are equivalent. The simplified formulas in bold are recommended for final calculation.
5.1 Standard Curve Plotting
Plot the standard concentration (x-axis) against ΔΔA_standard (y-axis) to generate the standard curve and obtain the standard equation. Substitute ΔΔA_test into the equation to obtain x (nmol/mL).
5.2 Glycerol Content Calculation
(1) Based on Protein Concentration
Glycerol (nmol/mg protein) = (Vsample × x) ÷ (Vsample × Cpr)= x ÷ Cpr
(2) Based on Sample Fresh Weight
Glycerol (nmol/g fresh weight) = (Vsample × x) ÷ (W × Vsample ÷ Vtotal sample)= x ÷ W
(3) Based on Cell Count
Glycerol (nmol/10⁶ cells) = (Vsample × x) ÷ (n × Vsample ÷ Vtotal sample)= x ÷ n
(4) Based on Liquid Volume
Glycerol (nmol/mL) = (Vsample × x) ÷ Vsample= x
Parameter Definitions:
Vsample: Volume of sample added to the reaction system (0.02 mL)
Vtotal sample: Volume of isopropanol added during extraction (1 mL)
Cpr: Sample protein concentration (mg/mL)
W: Sample weight (g)
n: Number of cells (in units of 10⁶)
6. Representative Results
The following data is for reference only. The experimenter needs to perform assays based on their specific samples.
Glycerol Standard Curve: y = 0.0003x - 0.0018, R² = 1
Figure 1: Glycerol Standard Curve
Precautions
1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
2. This product is for research use only. Not for use in clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
| G1505530 | 组件 | 48 T | 96 T | Storage |
| G1505530A | 试剂一 | 1EA (48T) | 1EA (96T) | -20℃. Store in the dark. |
| G1505530B | 试剂二 | 1EA (48T) | 1EA (96T) | 2-8℃. Store in the dark. |
| G1505530C | 试剂三 | 15 mL | 30 mL | 2-8℃. |
| G1505530D | 标准品 | 1 mL | 1 mL | 2-8℃. Store in the dark. |
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 |
|---|---|---|
 ZJ26F0333433 |
分析证书 | G1505530 |
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