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。
游离脂肪酸（FFA），又称非脂化脂肪酸（NEFA），主要由中性脂肪分解产生，是脂肪代谢过程中的中间产物，参与细胞增殖、炎症反应、激素调控等，也可作为一种具有多种生理功能的信号分子。游离脂肪酸与脂类代谢、糖代谢、内分泌功能密切相关，其含量是一项重要的生理生化指标，可作为评价和诊断疾病的辅助性参数，也可以反映食品贮藏过程中的品质变化。
检测原理：FFA与铜离子结合形成脂肪酸铜盐，并溶于氯仿；铜离子能够与显色液反应生成紫红色络合物，产物在550 nm处具有特征吸收峰，通过吸光值变化即可定量检测游离脂肪酸的含量。
检测范围：0.0313-2mM
灵敏度：0.0156mM
适用样本：血清（浆）、动植物组织、细胞、细菌
| A1492746 | Component | 48T | 96T | Storage |
| A1492746A | Cu Reagent | 6 mL | 12 mL | 2-8℃. Store in the dark. |
| A1492746B | Chromogen | 15 mL | 30 mL | 2-8℃. Store in the dark. |
| A1492746C | Standard (16.41 mg Palmitic Acid) | 1 EA | 1 EA | 2-8℃. Store in the dark. |
自备仪器和试剂
酶标仪或可见分光光度计（能测550nm处的吸光度）
恒温箱、制冰机、低温离心机
96孔板或微量玻璃比色皿、可调节式移液枪及枪头
匀浆器（如果是组织样本）
玻璃瓶（用于配制提取液）
正庚烷、无水甲醇、氯仿（三氯甲烷）
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Extraction Buffer（自备） | 取一个玻璃瓶，按照氯仿：正庚烷：无水甲醇=28:21:1的比例配制，盖紧后混匀 | 4℃避光保存 |
| Cu Reagent | 即用型；使用前平衡到室温；混匀后使用 | 4℃避光保存 |
| Chromogen | 即用型；使用前平衡到室温 | 4℃避光保存 |
| Standard | 临用前配制，加1mL Extraction Buffer，充分溶解得到64mM Standard | 未用完的已溶解的Standard可装于玻璃瓶中盖紧密封，4℃避光保存1个月 |
2、标准曲线设置
把64mM标准品按下表所示，用Extraction Buffer进行下一步稀释。
| 标准品序号 | 标准品 (μL) | Extraction Buffer体积 (μL) | 标准品浓度 (mM) |
| Std.1 | 20µL of 64mM | 620 | 2 |
| Std.2 | 100μL of Std.1 | 100 | 1 |
| Std.3 | 100μL of Std.2 | 100 | 0.5 |
| Std.4 | 100μL of Std.3 | 100 | 0.25 |
| Std.5 | 100μL of Std.4 | 100 | 0.125 |
| Std.6 | 100μL of Std.5 | 100 | 0.0625 |
| Std.7 | 100μL of Std.6 | 100 | 0.0313 |
注意：每次实验，请使用新稀释的标准品。
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6个月。
3.1 动物组织：称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8,000rpm，4℃离心10min，取上清液，置冰上待测。
3.2 植物组织：称取约0.1g样本，加入1mL Extraction Buffer捣碎，冰浴超声波破碎 5min（功率20%或200W，超声3s，间隔 7 s，重复30次），8,000rpm，4℃离心10min，取上清液，置冰上待测。
3.3 细胞或细菌：收集500万细胞或细菌到离心管内，用冷PBS清洗细胞，离心后弃上清，加入1mL Extraction Buffer，冰浴超声波破碎 5min（功率20%或200W，超声3s，间隔7s，重复30次），8,000rpm，4℃离心10min，取上清液，置冰上待测。
3.4 血清（浆）等液体：直接测定。
4、实验步骤
4.1 酶标仪或可见分光光度计预热30min以上，调节波长到550nm，可见分光光度计用去离子水调零。
4.2 样本测定（在EP管中依次加入下列试剂）：
| 试剂 | 空白管 (μL) | 标准管 (μL) | 测定管 (μL) |
| Extraction Buffer | 240 | 200 | 200 |
| 不同浓度 Std. | 0 | 40 | 0 |
| 样本 | 0 | 0 | 40 |
混匀后盖紧瓶盖，置于涡旋混合器上中速涡旋30s。
| Cu Reagent | 80 | 80 | 80 |
混匀后盖紧瓶盖，置于涡旋混合器上中速涡旋30s，室温（25℃）放置20min；2,000g，室温（25℃）离心5min。
| 上层溶液 | 50 | 50 | 50 |
| Chromogen | 200 | 200 | 200 |
4.3 室温（25℃）放置5min。每管取出200μL加入96孔板或微量玻璃比色皿对应的孔中，于550nm测定吸光值，计算ΔA测 =A测-A空 、ΔA标=A标-A空 （空白管只需做1管）。显色后务必在30min内测完。
注意：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A 测 大于酶标仪检测范围，样本可用Extraction Buffer进一步稀释，计算结果乘以稀释倍数。
5、结果计算
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
以标准液浓度为y轴，ΔA标为x轴绘制标准曲线（浓度为y轴更方便计算结果）。将ΔA测 带入标准曲线公式计算出y (mM)。
5.2 样本FFA含量计算
(1) 按样本质量计算
FFA含量 (μmol/g) =y×V提取÷W×n=y÷W×n
(2) 按细菌或细胞数量计算
FFA含量 (μmol/10⁴)=y÷(细胞或细菌数量÷V提取)×n=y÷500×n=0.002×y×n
(3) 按液体体积计算
FFA含量 (μmol/L)=1000×y×n
参数说明
V提取：加入提取液体积，1mL；
W：样本质量，g；
n：样本进一步稀释的稀释倍数；
500：细胞或细菌数量，以10⁴为单位；
1000：单位换算系数，1L=1000mL
6、结果展示
典型标准曲线：y=0.679x-0.0109，R²=0.9988
96孔板分析的游离脂肪酸 (FFA) 标准曲线。数据和曲线仅供参考，实验者需根据自己的实验建立标准曲线。
注意事项
1、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。
2、本产品仅供科学研究使用，不适用于临床诊断。
Free Fatty Acids (FFA), also known as Non-Esterified Fatty Acids (NEFA), are primarily produced by the hydrolysis of neutral fats. They are intermediate products in fat metabolism, involved in cell proliferation, inflammatory responses, and hormone regulation. FFA can also act as signaling molecules with various physiological functions. Free fatty acids are closely related to lipid metabolism, glucose metabolism, and endocrine function. Their concentration is an important physiological and biochemical indicator, serving as an auxiliary parameter for disease evaluation and diagnosis, and also reflecting quality changes during food storage.
Detection Principle: FFAs combine with copper ions to form copper soaps, which are soluble in chloroform. The copper ions can then react with a chromogenic solution to form a purplish-red complex. This product has a characteristic absorption peak at 550 nm. The FFA content can be quantified by measuring the change in absorbance.
Detection Range: 0.0313 - 2 mM
Sensitivity: 0.0156 mM
Applicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria.
| A1492746 | Component | 48T | 96T | Storage |
| A1492746A | Cu Reagent | 6 mL | 12 mL | 2-8℃. Store in the dark. |
| A1492746B | Chromogen | 15 mL | 30 mL | 2-8℃. Store in the dark. |
| A1492746C | Standard (16.41 mg Palmitic Acid) | 1 EA | 1 EA | 2-8℃. Store in the dark. |
User-Prepared Instruments and Reagents
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 550 nm)
Incubator, Ice maker, Low-temperature centrifuge
96-well plate or micro glass cuvettes, Adjustable pipettes and tips
Homogenizer (for tissue samples)
Glass bottle (for preparing extraction buffer)
n-Heptane, Anhydrous methanol, Chloroform
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Precautions |
| Extraction Buffer (Self-prepared) | In a glass bottle, mix Chloroform : n-Heptane : Anhydrous Methanol = 28 : 21 : 1. Cap tightly and mix well. | Store at 4°C protected from light. |
| Cu Reagent | Ready-to-use; equilibrate to room temperature before use; mix well before use. | Store at 4°C protected from light. |
| Chromogen | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Standard | Before use, dissolve contents in 1 mL of Extraction Buffer to obtain a 64 mM Standard solution. Mix well. | Unused dissolved Standard can be stored in a tightly sealed glass bottle at 4°C protected from light for 1 month. |
2. Standard Curve Setup
Dilute the 64 mM Standard further with Extraction Buffer as shown in the table below.
| Standard No. | Standard (μL) | Extraction Buffer Volume (μL) | Standard Concentration (mM) |
| Std.1 | 20µL of 64mM stock | 620 | 2 |
| Std.2 | 100μL of Std.1 | 100 | 1 |
| Std.3 | 100μL of Std.2 | 100 | 0.5 |
| Std.4 | 100μL of Std.3 | 100 | 0.25 |
| Std.5 | 100μL of Std.4 | 100 | 0.125 |
| Std.6 | 100μL of Std.5 | 100 | 0.0625 |
| Std.7 | 100μL of Std.6 | 100 | 0.0313 |
Note: Prepare freshly diluted standards for each experiment.
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 6 months.
3.1 Animal Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.3 Cells or Bacteria: Collect 5 million cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8,000 rpm, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.4 Serum (Plasma) and other liquids: Detect directly.
4. Assay Steps
4.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 550 nm. For spectrophotometers, zero the instrument with deionized water.
4.2 Sample Assay (Add reagents sequentially to EP tubes):
| Reagent | Blank Tube (μL) | Standard Tube (μL) | Test Tube (μL) |
| Extraction Buffer | 240 | 200 | 200 |
| Various Std. | 0 | 40 | 0 |
| Sample | 0 | 0 | 40 |
Cap the tubes tightly and vortex at medium speed for 30 seconds.
| Cu Reagent | 80 | 80 | 80 |
Cap the tubes tightly and vortex at medium speed for 30 seconds. Incubate at room temperature (25°C) for 20 minutes. Centrifuge at 2,000 g, room temperature (25°C) for 5 minutes.
| Upper Phase | 50 | 50 | 50 |
| Chromogen | 200 | 200 | 200 |
4.3 Incubate at room temperature (25°C) for 5 minutes. Transfer 200 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvettes. Measure the absorbance at 550 nm.
Calculate ΔAtest=Atest-Ablank and ΔAstd=Astd-Ablank (The blank tube only needs to be set up once).
Note: The measurement must be completed within 30 minutes after color development. It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If Atest exceeds the detection range of the instrument, dilute the sample further with Extraction Buffer and multiply the result by the dilution factor.
5. Result Calculation
We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas.
5.1 Standard Curve Plotting
Plot the standard curve with standard concentration as the y-axis and ΔAstd as the x-axis (using concentration as the y-axis facilitates calculation). Substitute ΔAtest into the standard curve equation to obtain y (mM).
5.2 Sample FFA Content Calculation
(1) Based on sample mass:
FFA Content (µmol/g fresh weight) = y × Vextract ÷ W × n = y ÷ W × n
(2) Based on bacterial or cell count:
FFA Content (µmol/10⁴ cells) = y ÷ (Cell or Bacterial Count ÷ Vextract ) × n = y ÷ 500 × n = 0.002 × y × n
(3) Based on liquid volume:
FFA Content (µmol/L) = 1000 × y × n
Parameter Description:
Vextract : Volume of Extraction Buffer added, 1 mL
W: Sample mass, g
n: Sample dilution factor (if further diluted)
500: Cell or bacterial count, in units of 10⁴
1000: Unit conversion factor, 1 L = 1000 mL
6. Result Presentation
Typical Standard Curve: y = 0.679x - 0.0109, R² = 0.9988
(Free Fatty Acid (FFA) standard curve analyzed using a 96-well plate. Data and curve are for reference only; users must establish their own standard curve based on their experiment.)
Precautions
1. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.
2. This product is for scientific research use only. Not intended for clinical diagnosis.
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 |
|---|---|---|
 ZJ25F1029029 |
分析证书 | A1492746 |
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