Oxidized Glutathione (GSSG) Content Assay Kit (DTNB, Colorimetric Method) - BioReagent, high purity

Cat. No.: O1505442

GRADE & PURITY BioReagent

Overview

Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamic acid, cysteine, and glycine. It is widely distributed in animal and plant tissues and microorganisms, helping maintain normal immune system function and possessing antioxidant and detoxification capabilities. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG, also known as glutathione disulfide, is formed by the oxidation of two glutathione molecules. GSSG can be reduced back to GSH by glutathione reductase. Consequently, the reduced form predominates in organisms. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing the cellular redox state.

Detection Principle: Endogenous GSH in the sample is masked by a GSH scavenger. Under the catalysis of glutathione reductase, GSSG is reduced to GSH. The newly generated GSH then reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce a yellow-colored 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. The GSSG content is quantitatively determined by measuring the change in absorbance.

Component	48T	Storage
Extraction Buffer	60 mL	2-8℃. Store in the dark.
Reagent 1	1EA	-20℃. Store in the dark.
Reagent 2	2EA	-20℃
Reagent 3	2 mL	2-8℃. Store in the dark.
Reagent 4	28 mL	2-8℃
Reagent 5	10 μL	-20℃
Standard	1EA	2-8℃

Reagent Preparation:

Reagent 1 (Liquid, 1 vial):

1.Before use, pipette 45 μL of Reagent 1 into a new microcentrifuge tube. Add 1.5 mL of ethanol and mix well for assay (prepare 2 tubes is sufficient).

2.The prepared solution can be stored for the duration of the kit's validity period.

Reagent 2 (Microcentrifuge tubes, 2 vials):

1.Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.

2.Add 0.6 mL of distilled water per vial to dissolve. Use after preparation.

3.Unused dissolved reagent can be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles. Use within one week.

Reagent 3 (Liquid, 1 bottle):

1.If solidified, incubate in a 25°C water bath briefly until completely melted before use.

2.The prepared solution can be stored for the duration of the kit's validity period.

Reagent 5 (Liquid, 1 vial):

1.Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the liquid at the bottom.

2.Add 1.1 mL of distilled water to dissolve. Use after preparation.

3.The prepared solution can be stored for the duration of the kit's validity period.

Standard (Powder, 1 vial):

1.Use this reagent if a new standard curve needs to be prepared.

2.Prepare according to the standard curve preparation steps in the instructions.

3.Use the dissolved standard within one week.

User-Prepared Instruments and Reagents
Mortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 1 mL cuvette, Centrifuge tubes, Spectrophotometer, Distilled water (Deionized water or Ultrapure water are acceptable).

Experimental Procedure

It is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing 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 Buffer and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C for 15 minutes. Collect the supernatant and keep it on ice for assay.
Note: Depending on research needs, a tissue mass (g) to Extraction Buffer volume (mL) ratio of 1:10 can be used for extraction.

1.2 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Disrupt the bacteria or cells by sonication (power 300W, pulse 3s on, 7s off, total duration 3 min). Centrifuge at 12,000 rpm, 4°C for 15 minutes. Collect the supernatant and keep it on ice for assay.
Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1.

1.3 Liquid Samples
Assay directly. If turbid, centrifuge and use the supernatant for assay.

2. Assay Steps

2.1 Preheat the visible spectrophotometer for 30 minutes. Set the wavelength to 412 nm. Zero the instrument with distilled water.
2.2 Incubate all reagents in a 25°C water bath for 10 minutes before use. For batch analysis, Reagents 2, 3, and 4 can be pre-mixed in a ratio of 20:40:520. Use 580 μL of this mixture per assay.
2.3 Add reagents sequentially to a 1 mL glass cuvette:

Reagent	Volume (μL) - Test Tube
Sample	80
Reagent 1	40

Mix gently and incubate for 10 minutes.

Reagent 2	20
Reagent 3	40
Reagent 4	520
Reagent 5	20

Mix thoroughly. At room temperature (25°C), immediately read the absorbance at 412 nm (A₁). Read the absorbance again after 25 minutes (A₂). Calculate ΔA = A₂ - A₁.

Note:
(1) If ΔA is close to zero, increase the sample volume (e.g., to 160 μL), correspondingly decreasing the volume of Reagent 4 to maintain a total reaction volume of 720 μL.
(2) Strictly control the reaction time, reading A₂ exactly at 25 minutes.

3. Standard Curve Preparation

3.1 Dissolve the Standard in 1.06 mL of distilled water (the stock solution must be used within two days and stored at -20°C). The concentration of the standard stock solution is 10 μmol/mL. Dilute the stock solution with distilled water to create six standard solutions with concentration gradients, e.g., 0, 1, 2, 3, 4, 5 nmol/mL. Standard concentrations can be adjusted based on actual sample levels.
3.2 Standard Dilution Reference Table:
Pipette 500 μL of the standard stock solution and add 500 μL of distilled water. Mix well to obtain a 5 μmol/mL standard dilution for use.

Standard Working Solution	Volume of Std. Dilution (μL)	Volume of Water (μL)	Concentration (nmol/mL)
Std.1	0	200	0
Std.2	40	160	1
Std.3	80	120	2
Std.4	120	80	3
Std.5	160	40	4
Std.5	200	0	5

Mix each standard tube well for use.
3.3 Operate according to the assay procedure for the Test tubes. Using the results, subtract the absorbance of the 0 concentration standard from the absorbance of each standard concentration to create a standard curve passing through the origin. 

Assay Table for Standard Curve:

Reagent (μL)	Standard Tube	0 Conc. Tube (once)
Standard	80
Distilled Water		80
Reagent 1	40	40

Mix gently and incubate for 10 minutes.

Reagent 2	20	20
Reagent 3	40	40
Reagent 4	520	520
Reagent 5	20	20

Mix thoroughly. At room temperature (25°C), immediately read the absorbance at 412 nm (A). Calculate ΔA = A_{Standard Tube} - A_{0 Concentration Tube}.

4. Calculation of Results

4.1 Standard Curve Equation: y = 1.0585x - 0.0077, R² = 0.9985; where x is the standard amount (nmol), and y is ΔA.

4.2 Sample GSSG Content Calculation

(1) Based on Sample Fresh Weight

Derived Formula: GSSG (nmol/g fresh weight) = (ΔA + 0.0077) ÷ 1.0585 ÷ (W × V₁ ÷ V)

Simplified Formula: GSSG (nmol/g fresh weight) = 11.8 × (ΔA + 0.0077) ÷ W

(2) Based on Cell Count

Derived Formula: GSSG (nmol/10⁴ cells) = (ΔA + 0.0077) ÷ 1.0585 ÷ (Cell Count × V₁ ÷ V)

Simplified Formula: GSSG (nmol/10⁴ cells) = 11.8 × (ΔA + 0.0077) ÷ Cell Count

(3) Based on Liquid Volume

Derived Formula: GSSG (nmol/mL) = (ΔA + 0.0077) ÷ 1.0585 ÷ V₁

Simplified Formula: GSSG (nmol/mL) = 11.8 × (ΔA + 0.0077)

Parameter Definitions:

V: Volume of Extraction Buffer added (1 mL)

V₁: Volume of sample added to the reaction (80 μL = 0.08 mL)

W: Sample weight (g)

Cell Count: Number of bacteria/cells (in 10⁴)

Precautions

1. The crude extract cannot be used for determining soluble protein content. If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.

2. Some reducing agents such as ascorbic acid, β-mercaptoethanol, dithiothreitol (DTT), and cysteine, or thiol-reactive compounds like maleimide compounds, can interfere with the glutathione assay. Therefore, the use of these substances during sample preparation should be avoided.

3. This product is for research use only. Not for use in clinical diagnosis.

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 6 months under corresponding storage conditions.