Reduced glutathione (GSH) detection kit (micro method)

Cat. No.: R1492762

GRADE & PURITY BioReagent

Descripción general

Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSH is the primary intracellular antioxidant thiol substance, playing important roles in antioxidation, protein thiol protection, and amino acid transmembrane transport. The ratio of reduced to oxidized glutathione (GSH/GSSG) can serve as a key dynamic indicator for assessing the cellular redox state.

Detection Principle: Reduced glutathione (GSH) reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce yellow-colored 5-thio-2-nitrobenzoic acid (TNB) and glutathione disulfide (GSSG). TNB has a characteristic absorption peak at 412 nm. The GSH content can be quantified by measuring the change in absorbance.

Detection Range: 2-400 µg/mL
Sensitivity: 2 µg/mL
Applicable Samples: Serum (plasma), animal/plant tissues, blood cells, cells, bacteria.

R1492762	Component	48T	96T	Storage
R1492762A	Extraction Buffer	70 mL	70 mL×2	2-8℃
R1492762B	Assay Buffer	10 mL	20 mL	2-8℃
R1492762C	Chromogen	4 mL	8 mL	2-8℃. Store in the dark.
R1492762D	Standard	1 EA	2 EA	2-8℃. Store in the dark.

Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.

User-Provided Instruments and Consumables

Type	Name	Notes
Instrument	Microplate Reader	Capable of measuring absorbance at 412 nm.
Consumables	96-well Microplate	Standard transparent plate.
Reagents	PBS (pH 7.4), Deionized Water	For standard and sample dilution.
Others	Homogenizer (for tissue samples), incubator, ice maker, low-temperature centrifuge, adjustable pipettes and tips	Using a multichannel pipette for large-scale detection can improve efficiency.

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	注意事项 Precautions
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Assay Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Chromogen	Ready-to-use	Protect from light during the experiment; store at 4°C in the dark.
Standard	Ready-to-use; equilibrate to room temperature before use.	Mass is 10 mg; aliquot and store at -20°C protected from light.

2. Standard Preparation

2.1 Dissolve the 10 mg standard in 1 mL of deionized water to prepare a 10 mg/mL stock solution. Mix well, equilibrate to room temperature. Aliquot and store at -20°C protected from light for up to 1 month.

2.2 Add 200 µL of Extraction Buffer to 1800 µL of deionized water to prepare the standard dilution buffer.

2.3 Use the standard dilution buffer to further dilute the stock solution to prepare standard working solutions with final concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 µg/mL.

Standard Working Solution	Standard (μL)	Standard Dilution Buffer (μL)	Concentration (µg/mL)
1	20 µL of 10 mg/mL stock	480	400
2	100 µL of 400 µg/mL sol (from 1)	100	200
3	100 µL of 200 µg/mL sol (from 2)	100	100
4	100 µL of 100 µg/mL sol (from 3)	100	50
5	100 µL of 50 µg/mL sol (from 4)	100	25
6	100 µL of 25 µg/mL sol (from 5)	100	12.5
7	100 µL of 12.5 µg/mL sol (from 6)	100	6.25

3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Processed samples must be assayed on the same day. Keep samples on ice during the assay procedure to prevent denaturation and inactivation.

3.1 Animal Tissues:
Weigh approximately 0.1 g of tissue, add 1 mL of pre-cooled Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant into a new tube and keep on ice for detection.

3.2 Plant Tissues:
Weigh approximately 0.1 g of plant tissue, add 1 mL of Extraction Buffer, and disrupt by ultrasound on ice (power 30% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.

3.3 Cells or Bacteria:
Collect 5×10⁶ cells or bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Add 1 mL of Extraction Buffer, and disrupt by ultrasound on ice (power 30% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.

3.4 Serum or Plasma:
Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Within 30 minutes, aspirate the upper plasma into another tube. Add an equal volume of Extraction Buffer, mix, then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.

3.5 Blood Cells:
Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Discard the upper plasma and wash the blood cells three times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600 g, 4°C for 10 min). Add an equal volume of Extraction Buffer, mix, and let stand at 4°C for 10 min. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.

4. Assay Steps
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 412 nm.
4.2 Assay System Setup: Perform the following operations in a 96-well plate. The standard curve and blank generally need to be performed only once.

Reagent	Blank Well (μL)	Standard Well (μL)	Test Well (μL)
Sample	0	0	20
Deionized Water	20	0	0
Standard Working Solution	0	20	0
Assay Buffer	140	140	140
Chromogen	40	40	40

4.3 Mix thoroughly. Incubate the reaction system at room temperature protected from light for 2 minutes.
4.4 Absorbance Measurement: Read the absorbance at 412 nm, recorded as A _blank , A _standard , and A _test .

5. Result Calculation
The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.

5.1 Data Processing
Calculate ΔA _standard = A _standard - A _blank , ΔA _test = A _test - A _blank .

5.2 Standard Curve Plotting
Plot the standard curve with standard concentration as the y-axis and ΔA _standard as the x-axis. Substitute ΔA _test into the equation to obtain the y value (µg/mL).

5.3 Sample GSH Content Calculation

(1) Based on sample mass:
GSH Content (µg/g fresh weight) = y × V _sample ÷ (W × V _sample ÷ V _total ) × n = y ÷ W × n

(2) Based on cell or bacterial count:
GSH Content (µg/10⁴ cells) = y ÷ (500 ÷ V _total ) × n = y ÷ 500 × n

(3) Based on liquid volume:
GSH Content (µg/mL) = y × 2 × n

(4) Based on protein concentration:
GSH Content (µg/mg prot) = y × V _sample ÷ (V _sample × Cpr) × n = y ÷ Cpr × n

Parameter Description:

• V _sample : Sample volume added, 20 µL;

• n: Sample dilution factor;

• Cpr: Sample protein concentration, mg/mL;

• W: Sample mass, g;

• V _total : Total volume of sample extract, 1 mL;

• 500: Cell or bacterial count, in units of 10⁴;

• 2: Dilution factor (for liquid samples, diluted 1:1 with Extraction Buffer).

6. Result Presentation
Typical Standard Curve: y = 342.21x + 1.4327, R² = 0.995

Example-1: 20 μL of mouse plasma was taken and assayed according to the procedure using a 96-well plate. The measured ΔA _test = A _{test - A blank = 0.155 - 0.084 = 0.071. Substituting into the standard curve, y = 25.73 µg/mL. Calculated based on liquid volume: GSH Content (µg/mL) = y × 2 × n = 51.46 µg/mL.}

Precautions

1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

2. The samples extracted with this kit are also suitable for the detection of oxidized glutathione (GSSG). Because the extraction buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of the extraction buffer. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.

4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.

5. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.

6. This product is for scientific research use only. Not intended for clinical diagnosis.

Frequently Asked Questions
Q: What should I do if the sample ΔA _test is too high or too low?
A: If the sample ΔA _test is >1.2, the GSH content in the sample is too high. Dilute the sample appropriately with Extraction Buffer (multiply by the dilution factor in the calculation). If the sample ΔA _test is <0.02, increase the sample amount.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light

Enviado en

Wet ice

Estabilidad y almacenamiento

Store at 2-8℃ long term (12 months). Store in the dark.