Reduced Glutathione (GSH) Content Assay Kit (DTNB, Colorimetric Method) - BioReagent, high purity

Cat. No.: R1505409

GRADE & PURITY BioReagent

Overview

Glutathione is a tripeptide containing a γ‑amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely distributed in animal and plant tissues as well as microorganisms. In living organisms, it helps maintain normal immune function and possesses antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSH is the major antioxidant thiol compound within cells and plays important roles in antioxidation, protection of protein thiol groups, and transmembrane amino acid transport. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing cellular redox status.

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

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 412 nm)
• Mortar (homogenizer), ice bucket (ice maker), tabletop centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath)
• 1 mL cuvettes, centrifuge tubes
• Distilled water (deionized or ultrapure water acceptable)

Experimental Workflow

It is recommended to first perform a pilot experiment using 1–3 samples with large differences (e.g., different types or groups) to familiarize yourself with the procedure and determine or adjust sample concentrations based on the pilot results, to avoid unnecessary waste of samples or reagents.

1. Sample Extraction

1.1 Tissue Samples

Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Solution, and homogenize on ice. Centrifuge at 4°C, 12,000 rpm for 15 minutes. Collect the supernatant and keep on ice for assay.

Notes:

(1) Depending on laboratory conditions, samples may first be ground in liquid nitrogen before adding Extraction Solution and homogenizing on ice.

(2) According to research needs, extraction can be performed at a tissue mass (g) to Extraction Solution volume (mL) ratio of 1:10.

1.2 Bacterial/Cell Samples

Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take 5 million bacteria or cells and add 1 mL of Extraction Solution. Sonicate to disrupt the bacteria/cells (power 300 W, pulse 3 s, interval 7 s, total time 3 minutes). Centrifuge at 4°C, 12,000 rpm for 15 minutes. Collect the supernatant and keep on ice for assay.

Note: If increasing sample amount, extract at a ratio of bacteria/cell count (10⁴ cells) to Extraction Solution (mL) of 500–1000:1.

1.3 Liquid Samples

Assay directly. If turbid, centrifuge and use the supernatant.

2. Assay Procedure

2.1 Preheat the visible spectrophotometer for 30 minutes, set wavelength to 412 nm, and zero with distilled water.

2.2 Warm all reagents in a 25°C water bath for 10 minutes before use.

2.3 In a 1 mL glass cuvette (1 cm path length), add in order:

Reagent (μL)	Test Tube	Control Tube
Sample	80	80
Reagent 1	480	640
Reagent 2	160	/

Mix immediately, let stand for 5 minutes, then read absorbance (A) at 412 nm. ΔA = A_Test – A_Control.

Notes:

(1) For the same batch of samples, one sample control tube is sufficient.

(2) If ΔA is near zero, increase the sample amount (e.g., to 0.2 g tissue) or increase the sample volume added (e.g., to 160 μL, with Reagent 1 reduced accordingly).

2. Standard Curve Preparation

3.1 Dissolve the Standard in 2 mL of distilled water (the stock solution should be used within two days and stored at –20°C). The stock concentration is 1 μmol/mL. Dilute the stock with distilled water to create six concentration gradients, e.g., 0, 0.2, 0.4, 0.6, 0.8, 1.0 μmol/mL. Adjust standard concentrations according to actual sample levels.

3.2 Standard dilution reference table:

Standard Working Solution	Standard Stock (μL)	Water (μL)	Concentration (μmol/mL)
Std.1	0	200	0
Std. 2	40	160	0.2
Std. 3	80	120	0.4
Std. 4	120	80	0.6
Std. 5	160	40	0.8
Std. 6	200	0	1.0

Note: Mix each standard tube well before use.

3.3 Follow the addition table for the test tubes. Using the results, subtract the absorbance of the 0‑concentration tube from each standard absorbance and construct a standard curve.

Reagent (μL)	Standard Tube	0‑concentration Tube (once only)
Standard	80	/
Distilled water	/	80
Reagent 1	480	480
Reagent 2	160	160

Mix immediately, let stand for 5 minutes, then read absorbance (A) at 412 nm. ΔA = A_Test – A_0‑conc.

4. Result Calculation

4.1 Standard curve: y = 1.5674x – 0.015, R² = 0.9993; where x is standard concentration (μmol/mL), y is ΔA.

4.2 Sample GSH Content Calculation

(1) Based on Protein Concentration

GSH (μmol/mg prot) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (V₁ × Cpr) = 0.638 × (ΔA + 0.015) ÷ Cpr

(2) Based on sample fresh weight:

GSH (μmol/g fresh weight) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (W × V₁ ÷ V) = 0.638 × (ΔA + 0.015) ÷ W

(3) Based on cell count:

GSH (μmol/10⁴ cells) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (500 × V₁ ÷ V) = 0.638 × (ΔA + 0.015) ÷ 500

(4) Based on Liquid Volume

GSH (μmol/mL) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ V₁ = 0.638 × (ΔA + 0.015)

Parameter Explanation:

• V: Total volume of supernatant (1 mL)

• V₁: Volume of supernatant added to the reaction system (80 μL = 0.08 mL)

• W: Sample mass (g)

• 500: Bacteria/cell count, in 10⁴ units

• GSH Molecular Weight: 307.3

• Cpr: Protein concentration (mg/mL)

Precautions

1. The supernatant cannot be used for protein concentration determination. If protein content needs to be measured, take a separate aliquot of the same sample. For protein concentration determination, Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready‑to‑Use BCA Protein Quantification Kit is recommended.

2. Biochemical assay reagents are generally irritating and biologically toxic. For your safety and health, maintain appropriate biosafety precautions throughout the experiment: wear lab coats, masks, gloves, head covers, etc., and perform experiments in a fume hood or biosafety cabinet.

3. This product is for scientific research only and not suitable for clinical diagnosis.

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

R1505409	Component	48T	Storage
R1505409A	Extraction Solution	50 mL	2-8℃. Store in the dark.
R1505409B	Reagent 1	60 mL	2-8℃.
R1505409C	Reagent 2	8 mL	2-8℃. Store in the dark.
R1505409D	Standard	1 EA	2-8℃.

Notes:

(1) Reagent 2: If solidified, incubate in a 25°C water bath briefly until fully melted before use.

(2) Standard: Required for constructing a new standard curve. Prepare according to the instructions in the manual. Use the dissolved standard within one week.