γ-Aminobutyric Acid (GABA) Content Assay Kit (Phenol-Sodium Hypochlorite, Colorimetric Method)

Cat. No.: A1515961

GRADE & PURITY BioReagent

Overview

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:

Item No.	Appearance	Components	50T	Storage
A1515961A	Liquid	Extraction Solution: Liquid	60 mL	2-8℃.
A1515961B	Liquid	Reagent 1: Liquid	10 mL	2-8℃.
A1515961C	Liquid	Reagent 2: Liquid	20 mL	2-8℃. Store in the dark.
A1515961D	Liquid	Reagent 3: Liquid	20 mL	2-8℃. Store in the dark.
A1515961E	Liquid	Reagent 4: Liquid	6mL	2-8℃.
A1515961F	Solid	Standard: Powder	1 vial	2-8℃.

Standard Reference (for standard curve preparation): Before use, add 1 mL of distilled water and dissolve thoroughly to obtain a 10 mg/mL γ-aminobutyric acid solution. Serially dilute with distilled water. Stable for 2 weeks at 4 °C. (Optional)

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.

Reagent (µL)	Test Tube	Blank Tube
Test sample	100	/
Distilled water	/	100
Reagent 1	50	50
Reagent 2	400	400
Reagent 3	350	350
Reagent 4	100	100

1. After adding Reagent 2, mix thoroughly and incubate at room temperature for 5 min.
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:A_test and A_blank. Calculate ΔA = A_test − A_blank. 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 ×V_sample ÷ V_sample = 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_sample ÷ V_sample ÷ 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 × VV_sample ÷ (W × V_sample ÷ V_{total 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_{total sample} ÷ 500 = 0.857 × (ΔA − 0.0174) ÷ 500

V_sample: Volume of sample added to reaction system, 0.1 mL;

V_{total sample}: 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_test = 0.235, A_blank = 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.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (6 months). Store in the dark.

Contents & Storage

Item No.	Appearance	Components	50T	Storage
A1515961A	Liquid	Extraction Solution: Liquid	60 mL	2-8℃.
A1515961B	Liquid	Reagent 1: Liquid	10 mL	2-8℃.
A1515961C	Liquid	Reagent 2: Liquid	10 mL	2-8℃. Store in the dark.
A1515961D	Liquid	Reagent 3: Liquid	10 mL	2-8℃.
A1515961E	Liquid	Reagent 4: Liquid	50 mL	2-8℃.
A1515961F	Solid	Standard: Powder	1 vial	2-8℃.

Standard Reference (for standard curve preparation): Before use, add 1 mL of distilled water and dissolve thoroughly to obtain a 10 mg/mL γ-aminobutyric acid solution. Serially dilute with distilled water. Stable for 2 weeks at 4 °C. (Optional)