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γ-氨基丁酸（γ-aminobutyric acid, 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，主要在抵御低温、干旱、虫害等逆境胁迫、诱导乙烯合成、调节生长发育等方面发挥作用。 在哺乳动物脑内，GABA是一种有效的抑制性神经递质，对于改善脑机能、增进肝肾功能等方面有辅助治疗的作用，目前在医药和保健食品中已有广泛的应用。
测定原理：
GABA与次氯酸盐、酚类反应，生成的蓝绿色物质在640nm处有特征吸收峰，通过检测640nm处的吸光值，可计算得到样本GABA含量。
注：
1. 正式测定前务必取2-3个预期差异较大的样本做预测定
2. 本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
组分与储存：
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自备仪器和试剂
样本处理：
1.组织：按照组织质量（g)：提取液体积（mL）为1：5-10的比例（建议称取0.1g组织，加入1mL提取液）处理样品，冰浴匀浆，95℃处理2h（密封以防止水分散失），期间振荡混匀3-5次，冷却至室温，8000g常温离心10min，取上清液即为待测样本。
2. 细菌、真菌：按照细胞数量（10⁴个）：提取液体积（mL）为500~1000：1的比例（建议500万细胞加入1mL提取液），冰浴超声破碎（功率200W，超声3s，间隔10s，重复30次)，95℃处理2h（密封以防止水分散失)，期间振荡混匀3-5次，冷却至室温，8000g常温离心10min，取上清液即为待测样本。
3.液体样本：澄清的液体样本直接测定，若浑浊则离心后取上清检测。
测定步骤：
分光光度计预热 30 min 以上，调节波长至 640 nm，蒸馏水调零。
2. 试剂三加入后，充分混匀，95℃水浴10 min，冷却至室温。 3. 试剂四加入后，混匀，将反应液转移至1mL玻璃比色皿，测定640nm处吸光值 |
γ-氨基丁酸含量计算公式：
标准条件下测定的回归方程为y=1.167x+0.0174，R²=0.9981，x为标准品浓度（mg/mL），y为吸光值。
γ-氨基丁酸含量
(3) 按细菌或细胞密度计算：
GABA含量(mg/10⁴ cell)＝(ΔA-0.0174)÷1.167×V样总÷500=0.857×(ΔA-0.0174)÷500
V样：反应样本体积，0.1mL；
V样总：加入提取液体积，1mL；
Cpr：样本蛋白质浓度mg/mL；
W：样本质量，g；
500：细菌或细胞总数，500万。
注意事项：
1. ΔA线性范围为0.02-1.1，可测定γ-氨基丁酸浓度范围0.02-1mg/mL；如果样本ΔA大于1.1，建议稀释待测样本后重新测定（计算公式中乘以相应稀释倍数），如果ΔA小于0.02，建议适当增加样本量。
2. 95℃处理过程中应注意密封以防止水分散失，推荐使用螺纹盖离心管或冻存管。
3. 提取液中含有蛋白去除组分，待测样本不能用于蛋白浓度测定，若需要使用蛋白浓度计算结果，需另取样本使用PBS或生理盐水按照相同比例，单独提取后再进行蛋白浓度测定；
4. 检测样本中应不含有谷氨酰胺，且氨基酸含量应低于10mg/g或1mg/mL。
实验案例：
称取0.1055g紫叶李幼嫩叶片，按步骤对样本进行处理，结果：A测定=0.235，A空白=0.036，ΔA=0.199，紫叶李叶片中γ-氨基丁酸含量为1.617mg/g。
常见问题及解决方案：
1. 样品读数重复性差
① 样品类型不合适：参考说明书了解不兼容的样品类型。
② 组织样品匀浆不充分：延长超声时间/增加匀浆器的研磨次数。
③ 样品反复冻融次数过多：将样品分装，不可反复冻融。
④ 样品中含有干扰物质：排查干扰物质，必要时对样品进行脱蛋白处理。
⑤ 样品读数超出线性范围：对样品进行浓缩或稀释，使读数处于线性范围内。
⑥ 孔中有气泡：气泡会干扰读数，操作时尽量避免产生气泡，读数前务必去除气泡。
2. 样品和标准品读数偏低/偏高
① 样品放置时间过长或储存不当：使用新鲜制备的样品，按推荐温度保存至使用前。
② 孵育时间/温度不当：参考说明书推荐的孵育时间和/或温度。
③ 试剂加样量错误：检查移液枪是否校准（始终使用能吸取整体积的最小量程移液枪）；尽量避免吸取过小体积的试剂。
γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid widely distributed in animals, plants and microorganisms. In plants, GABA is mainly synthesized via the decarboxylation of L-glutamate catalyzed by glutamate decarboxylase. It plays vital roles in resisting abiotic and biotic stresses such as low temperature, drought and pest infestation, stimulating ethylene biosynthesis, and regulating plant growth and development. In the mammalian brain, GABA acts as a potent inhibitory neurotransmitter. It exhibits adjuvant therapeutic effects in improving brain function and protecting liver and kidney functions, and has been extensively applied in pharmaceuticals and health food products.
Assay Principle:
GABA reacts with hypochlorite and phenols to form a blue-green complex with a characteristic absorption peak at 640 nm. The GABA content in samples can be quantified by measuring the absorbance at 640 nm.
Notes:
1. A preliminary test must be performed with 2–3 samples featuring significantly different expected GABA concentrations prior to formal detection.
2. This kit is for research use only; it shall not be used for clinical diagnosis or any other purposes.
Contents & Storage:
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Self-prepared Instruments and Reagents:
⁕Spectrophotometer (capable of absorbance measurement at 640 nm)
⁕Electronic balance, tabletop centrifuge, water bath, pipettes, mortar, shaker, 1 mL glass cuvettes
⁕Ice, distilled water
Sample Pretreatment:
1. Tissue samples: Prepare samples at a ratio of tissue mass (g) to extractant volume (mL) of 1:5–10 (0.1 g tissue with 1 mL extractant is recommended). Homogenize on ice, then incubate at 95°C for 2 h in a sealed container to avoid water loss. Vortex and mix thoroughly 3–5 times during incubation. Cool to room temperature, centrifuge at 8000 g for 10 min at ambient temperature, and collect the supernatant as the test sample.
2. Bacteria and fungi samples: Prepare samples at a ratio of cell count (10⁴ cells) to extractant volume (mL) of 500–1000:1 (5 × 10⁶ cells with 1 mL extractant is recommended). Lyse cells by sonication on ice (power: 200 W; sonication time: 3 s; interval: 10 s; 30 cycles total). Incubate at 95 °C for 2 h in a sealed container to prevent water evaporation. Vortex and mix thoroughly 3–5 times during incubation. Cool to room temperature, centrifuge at 8000 g for 10 min at ambient temperature, and collect the supernatant as the test sample.
3. Liquid samples: Clear liquid samples can be tested directly. Turbid samples should be centrifuged, and the supernatant is collected for detection.
Assay Procedures:
Preheat the spectrophotometer for more than 30 min, set the wavelength to 640 nm, and zero the instrument with distilled water.
2. After adding Reagent 3, mix thoroughly, incubate in a 95 °C water bath for 10 min, then cool to room temperature. 3. After adding Reagent 4, mix the solution well, transfer the reaction mixture into a 1 mL glass cuvette, and measure the absorbance values at 640 nm:Atest and Ablank. Calculate ΔA = Atest − Ablank. Note: The blank tube only needs to be measured 1–2 times. |
Calculation Formula for γ-Aminobutyric Acid Content:
The regression equation obtained under standard testing conditions is y = 1.167x + 0.0174, with R² = 0.9981. Where x=standard substance concentration (mg/mL), y=absorbance value.
γ-Aminobutyric Acid Content
1. Calculation of GABA content in liquid samples:
GABA content (mg/mL) = (ΔA − 0.0174) ÷ 1.167 ×Vsample ÷ Vsample = 0.857 × (ΔA − 0.0174)
2. Calculation of GABA content in tissues, bacteria or cells:
(1) Calculation based on sample protein concentration:
GABA content (mg/mg prot) = (ΔA − 0.0174) ÷ 1.167 × V<sub>sample</sub> ÷ V<sub>sample</sub> ÷ Cpr = 0.857 × (ΔA − 0.0174) ÷ Cpr
Protein concentration needs to be measured separately. It is recommended to use the BCA Protein Concentration Assay Kit produced by our company.
(2) Calculation based on sample fresh weight:
GABA content (mg/g) = (ΔA − 0.0174) ÷ 1.167 × VVsample ÷ (W × Vsample ÷ Vtotal sample) = 0.857 × (ΔA − 0.0174) ÷ W
(3) Calculation based on bacterial or cell density:
GABA content (mg/10⁴ cells) = (ΔA − 0.0174) ÷ 1.167 × V<sub>total sample</sub> ÷ 500 = 0.857 × (ΔA − 0.0174) ÷ 500
V<sub>sample</sub>: Volume of sample added to reaction system, 0.1 mL;
V<sub>total sample</sub>: Total volume of extractant added, 1 mL;
Cpr: Sample protein concentration, mg/mL;
W: Mass of sample, g;
500: Total number of bacteria or cells, 5 million.
Precautions:
1. The linear range of ΔA is 0.02–1.1, corresponding to a measurable γ-aminobutyric acid concentration range of 0.02–1 mg/mL. If the ΔA value of the sample exceeds 1.1, dilute the test sample and re-measure (multiply the final result by the corresponding dilution factor in the calculation formula). If ΔA is less than 0.02, appropriately increase the sample dosage.
2. Ensure tight sealing during the 95 °C incubation to prevent water loss; screw-cap centrifuge tubes or cryotubes are recommended.
3. The extract contains protein removal components, so the treated test sample cannot be used for protein concentration determination. If results normalized by protein content are required, take a separate aliquot of sample, extract it independently with PBS or normal saline at the same ratio, and then measure the protein concentration.
4. Test samples must be free of glutamine, and the total amino acid content shall not exceed 10 mg/g or 1 mg/mL.
Experimental Case:
0.1055 g of young Prunus cerasifera leaves were weighed and processed following the procedures. Measured absorbance values: A<sub>test</sub> = 0.235, A<sub>blank</sub> = 0.036, ΔA = 0.199. The γ-aminobutyric acid content in Prunus cerasifera leaves was calculated as 1.617 mg/g.
Frequently Encountered Problems and Troubleshooting:
1. Poor repeatability of sample absorbance readings
① Incompatible sample matrix: Refer to the manual for incompatible sample types.
② Incomplete homogenization of tissue samples: Extend sonication duration or increase homogenization cycles.
③ Excessive freeze-thaw cycles: Aliquot samples to avoid repeated freezing and thawing.
④ Presence of interfering substances in samples: Identify interfering components; perform deproteinization if necessary.
⑤ Readings outside the linear range: Concentrate or dilute samples to bring absorbance values within the linear range.
⑥ Air bubbles in cuvettes: Bubbles interfere with photometric readings. Minimize bubble formation during operation and remove all bubbles before measurement.
2. Abnormally low or high absorbance for samples and standards
① Prolonged sample storage or improper preservation: Use freshly prepared samples and store at the recommended temperature until use.
② Incorrect incubation time or temperature: Follow the incubation time and temperature specified in the manual.
③ Inaccurate reagent pipetting: Verify pipette calibration (always use the smallest-volume pipette capable of dispensing the target volume); avoid pipetting excessively small volumes of reagents.
| 项目号 | 物理外观 | 组件 | 50T | 储存 |
| A1515961A | 液体 | 提取液：液体 | 60 mL | 2-8℃. |
| A1515961B | 液体 | 试剂一：液体 | 10 mL | 2-8℃. |
| A1515961C | 液体 | 试剂二：液体 | 10 mL | 2-8℃，避光 |
| A1515961D | 液体 | 试剂三：液体 | 10 mL | 2-8℃. |
| A1515961E | 液体 | 试剂四：液体 | 50 mL | 2-8℃. |
A1515961F | 固体 | 标准品：粉剂 | 1支 | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636889 |
分析证书 | A1515961 |
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