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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。
γ-氨基丁酸（GABA）是一种四碳非蛋白质组成的氨基酸，广泛存在于动植物和微生物体内。在植物体内，GABA主要由谷氨酸脱羧酶催化 L-谷氨酸脱羧生成。在哺乳动物脑内，GABA 是一种有效的抑制性神经递质，具有降血压、增进脑活力、营养神经细胞、保持神经安定、促进生长激素分泌和保肝利肾等作用，目前在医药和保健食品中已有广泛的应用。
本试剂盒检测原理如下：苯酚和次氯酸钠与GABA 反应，产生蓝绿色产物，在640 nm 有最大吸光值。
产品组分
A1515841 | 组件 | 物理外观 | 100T | 储存 | 单次测试使用量 |
A1515841A | 提取液 | 液体 | 50 mL | 2-8℃ | 按需 |
A1515841B | 缓冲液 | 液体 | 5 mL | 2-8℃ | 50 μL |
A1515841C | 显色剂A | 液体 | 10 mL | 2-8℃，避光 | 100 μL |
A1515841D | 显色剂B | 液体 | 4 mL | 2-8℃，避光 | 40 μL |
A1515841E | 标准品（10 μmol/mL） | 液体 | 1 mL | 2-8℃ | 按需 |
注：显色剂A、显色剂B有毒且有刺激性气味，建议在通风橱进行实验。 |
使用方法
1. 需自备试剂
60%乙醇。
2. 样本制备
称取约0.05 g 样本，加入0.5 mL 提取液，充分匀浆，转移至EP 管，95℃水浴2 h（盖紧，在管口缠绕封口膜，以防止水分散失）。8000 × g ，25℃离心10 min，取上清待测。
3. 标准品的配制
按照下表用提取液将标准品（10 μmol/mL）稀释至0、1.0、2.0、4.0、5.0、7.0、9.0、10 μmol/mL。
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
浓度（μmol/mL） | 0 | 1.0 | 2.0 | 4.0 | 5.0 | 7.0 | 9.0 | 10.0 |
标准品（μL） | 0 | 20 | 40 | 80 | 100 | 140 | 180 | 200 |
提取液（μL） | 200 | 180 | 160 | 120 | 100 | 60 | 20 | 0 |
4. 样本测定
按照下表在1.5mL EP管中进行检测体系配制，每个样品都需要设置一个对照管，标准管一般只做1 次。
| 标准孔 | 测定孔 |
待测样本（μL） | - | 30 |
不同浓度标准品（μL） | 30 | - |
缓冲液（μL） | 50 | 50 |
显色剂A（μL） | 100 | 100 |
混匀，室温静置5min。 | ||
显色剂B（μL） | 40 | 40 |
混匀，95℃ 水浴加热10 min，冰浴冷却。 | ||
60%乙醇（μL） | 200 | 200 |
混匀，各取200 μL于96 孔板中测定640 nm 处OD 值。 |
5. 结果计算
标准品拟合曲线：y = ax + b
GABA含量计算公式：GABA 含量(μmol/g wet weight)= (ΔA-b) ÷ a ×V ÷ m× f
y：标准品OD 值-空白OD 值(标准品浓度为0 时的OD 值)；
x：标准品的浓度；
a：标曲的斜率；
b：标曲的截距；
ΔA：样本OD 值-空白OD 值(标准浓度为0 时的OD 值)；
m：组织湿重质量，g；
V：加入提取液的体积，mL；
f：样本加入检测体系前的稀释倍数
注意事项
1. 显色剂A、显色剂B有毒且有刺激性气味，建议在通风橱进行实验。
2. 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。
3. 为了您的安全和健康，请穿实验服并戴丁腈手套操作，请勿使用乳胶手套。
γ-aminobutyric acid (GABA) is a four-carbon, non-proteinogenic amino acid widely present in animals, plants, and microorganisms. In plants, GABA is primarily produced by the decarboxylation of L-glutamate catalyzed by glutamate decarboxylase. In mammalian brains, GABA acts as an effective inhibitory neurotransmitter, exerting effects such as lowering blood pressure, enhancing brain function, nourishing nerve cells, maintaining neural stability, promoting growth hormone secretion, and protecting the liver and kidneys. It has been widely applied in pharmaceuticals and health supplements.
The detection principle of this kit is as follows: phenol and sodium hypochlorite react with GABA to produce a blue-green compound that shows maximum absorbance at 640 nm.
Kit Contents
A1515841 | Components | Appearance | 100T | Storage | Quantity Per Test |
A1515841A | Extraction Buffer | Liquid | 50 mL | 2-8℃. | As needed |
A1515841B | Buffer Solution | Liquid | 5 mL | 2-8℃. | 50 μL |
A1515841C | Chromogenic Agent A | Liquid | 10 mL | 2-8℃. Store in the dark. | 100 μL |
A1515841D | Chromogenic Agent B | Liquid | 4 mL | 2-8℃. Store in the dark. | 40 μL |
A1515841E | Standard Solution (10 μmol/mL) | Liquid | 1 mL | 2-8℃. | As needed |
Note: Both Chromogenic Agent A and Chromogenic Agent B are toxic and have a pungent odor; it is recommended to conduct the experiment in a fume hood. |
Instructions for Use
1. Reagents to be prepared by yourself
60% ethanol.
2. Sample preparation
Weigh approximately 0.05 g of the sample, add 0.5 mL of the extraction solution, thoroughly homogenize, transfer to an EP tube, incubate at 95℃ in a water bath for 2 hours (cover tightly and wrap a sealing film around the tube opening to prevent water loss). Centrifuge at 8000 × g for 10 minutes at 25℃, then take the supernatant for testing.
3. Preparation of standards
Use the Extraction Buffer to dilute the standard (10 μmol/mL) to 0, 1.0, 2.0, 4.0, 5.0, 7.0, 9.0, and 10 μmol/mL according to the table below.
Serial Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
Concentration (μmol/mL) | 0 | 1.0 | 2.0 | 4.0 | 5.0 | 7.0 | 9.0 | 10.0 |
Standard solution (μL) | 0 | 20 | 40 | 80 | 100 | 140 | 180 | 200 |
Extraction Buffer (μL) | 200 | 180 | 160 | 120 | 100 | 60 | 20 | 0 |
4. Sample Measurement
According to the table below, prepare the detection system in a 1.5 mL EP tube. For each sample, a control tube needs to be set up. The standard tube is generally only prepared once.
Reagent name | Standard well | Sample well |
Sample (μL) | - | 50 |
Standard solutions of different concentrations (μL) | 50 | - |
Buffer (μL) | 50 | 50 |
Color developer A (μL) | 100 | 100 |
Mix well and let it stand at room temperature for 5 minutes. | ||
Color developer B (μL) | 40 | 40 |
Mix well, heat in a water bath at 95°C for 10 minutes, then cool under ice. | ||
60% ethanol (μL) | 200 | 200 |
Mix well, take 200 μL from each and measure the OD value at 640 nm in a 96-well plate. |
5. Result calculation
Standard curve equation: y = ax + b
The formula for calculating GABA content: GABA content (μmol/g wet weight) = ((ΔA - b) ÷ a) × V ÷ m × f
y: measured OD value minus blank OD value (OD at zero standard concentration);
x: standard concentration;
a: slope of the standard curve;
b: intercept of the standard curve;
ΔA: Sample OD value − blank OD value (OD value at standard concentration = 0);
m: wet weight of the sample, g;
V: volume of the extraction solution added, mL;
f: dilution factor applied to the sample prior to addition into the assay system.
Precautions
1. Both Chromogenic Agent A and Chromogenic Agent B are toxic and have a pungent odor; it is recommended to conduct the experiment in a fume hood.
2. Before the formal test, it is recommended to select 2-3 samples with significant expected differences for a preliminary experiment.
3. For your safety and health, please wear a laboratory coat and latex gloves during the operation. Do not use latex gloves.
A1515841 | 组件 | 物理外观 | 100T | 储存 | 单次测试使用量 |
A1515841A | 提取液 | 液体 | 50 mL | 2-8℃. | 按需 |
A1515841B | 缓冲液 | 液体 | 5 mL | 2-8℃. | 50 μL |
A1515841C | 显色剂A | 液体 | 10 mL | 2-8℃，避光 | 100 μL |
A1515841D | 显色剂B | 液体 | 4 mL | 2-8℃，避光 | 40 μL |
A1515841E | 标准品（10 μmol/mL） | 液体 | 1 mL | 2-8℃. | 按需 |
注：显色剂A、显色剂B有毒且有刺激性气味，建议在通风橱进行实验。 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838528 |
分析证书 | A1515841 |
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