Total Glutathione (T-GSH) Assay Kit (for Biochemical Assay Kit)

Product Information

Product Description/Introduction

Glutathione (glutamyl-cysteinyl-glycine) is a small peptide composed of three amino acid residues, which is widely found in the body. Glutathione exists in two different forms, reduced glutathione (GSH) and oxidized glutathione (GSSG), and can be converted into each other under certain conditions. GSH is an important antioxidant in animal cells and is the main source of sulfhydryl groups in most cells. Under physiological conditions, GSSG is reduced by glutathione reductase, so GSH is the main form in the body.

This kit uses glutathione reductase to reduce GSSG to GSH, and then reacts GSH with DTNB to generate TNB and GSSG.The TNB produced is positively correlated with total glutathione, so the amount of total glutathione (T-GSH) can be assessed by measuring TNB production.GSSG is re-converted to GSH under the action of glutathione reductase, and then continues to react with DTNB in the cyclic system, which also greatly enhances the sensitivity of detection.

Storage and Shipping Conditions

Ship with wet ice; Store at -20 away from light for 6 months.

Product Content

Assay Protocol / Procedures

1. Sample Preparation:

1.1. Sample pre-treatment

1.1.1. Plasma and serum samples: add protein removal reagent at a ratio of 1 to 4, and mix thoroughly;Centrifuge at 10000g at 4 for 10-15min and take the supernatant for subsequent total glutathione detection.

1.1.2. Tissue samples: add protein removal reagent to the tissue for homogenization and lysis (weight g: volume mL is 1 to 9); after homogenization and lysis, centrifuge the tissue at 10,000 g for 10-15 min at 4°C, and remove the supernatant for subsequent total glutathione assay;

1.1.3. Cell samples: After cell collection, cells are resuspended using protein removal reagent, approximately 100-200 μL of reagent per 1 × 10⁷ cells, which are lysed by sonication or on ice; after treatment, centrifugation is performed at 4°C, 10,000 g for 10-15 min, and the supernatant is extracted for subsequent total glutathione assay;

1.2. Standard preparation: The protein removal reagent is mixed with ultrapure water at the ratio of 1:9, and the glutathione standard product (1mmol/L) is gradient diluted with it (for example, it can be set to 50/25/12.5/6.25/3.125μmol/L), and then used for data analysis by standard curve method after follow-up testing with the sample to be tested;Or appropriately diluted into a known concentration of the standard product, and the sample to be tested after follow-up testing, used for standard conversion method data analysis;

2. Total glutathione assay:

2.1. Glutathione reductase and total glutathione assay buffer are mixed 1:9 to configure a working solution for glutathione assay;

2.2. 1:19 mix of assay probe and ultrapure water to form assay probe working solution;

2.3. Refer to the table below for total glutathione assay;

3. Data analysis:

3.1. Standard curve method:

3.1.1. The standard curve is plotted according to the gradient dilution of the standard group value - blank group value data using Excel and other software: Y = a X + b (Y is the standard group value - blank group value; X is the total glutathione content; a is the slope of the standard curve; b is the intercept of the standard curve);

3.1.2. Calculation of the total glutathione content of the samples:

Sample total glutathione (μmol/L) = [(Sample group value - Blank group value) - b] / a × sample dilution

3.1.3. Calculation of total iron ion concentration in tissue and cell samples:

Total ferrous ion concentration per unit sample (μmol/gprot) = [(Sample group value - Blank group value) - b]/a × sample dilution / sample protein concentration (gprot/L)

3.2. Standard product conversion method:

3.2.1. Calculation of MDA concentration per unit serum plasma sample:

Sample total glutathione (μmol/L) = (sample group value - blank group value)/(standard group value - blank group value) × standard total glutathione content (μmol/L) × sample dilution

Note

1. Dissolve and gently shake the reagents before use to ensure that the components are homogeneous.

2. For first time use, the reagents can be stored in separate packages as appropriate to avoid repeated freezing and thawing which may affect the performance of the reagents.

3. Glutathione assay working solution and assay probe working solution are used as much as needed.

4. When analyzing the data by the standard conversion method, the concentration of the sample to be tested and the standard should be within the linear detection range of the kit; when analyzing the data by the standard curve method, make sure that the R² of the standard curve is more than 0.99.

5. Reducing agents as well as sulfhydryl-active compounds can severely interfere with the determination of this kit.

6. For your health and safety, please wear lab coat and gloves during operation.

Appendix: Technical parameters

For Research Use Only!

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