Reduced Glutathione (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 is based on the principle that reduced glutathione can react with DTNB to produce TNB and GSSG. TNB is a yellow product, and the GSH content of the sample can be effectively determined by measuring its absorbance at 412 nm.

2GSH+DTNB  GSSG+2TNB

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: mix the sample with Protein Removal Reagent 1:4 (e.g. 50 μL serum: 200 μL Protein Removal Reagent); Centrifuge at 10,000 g for 10-15 min at 4°C and remove the supernatant for subsequent reduced glutathione detection;

1.1.2. Tissue samples: Homogenization lysis was performed using a suitable buffer (PBS, saline, etc.), where a ratio of 1 to 9 (0.1 g: 0.9 mL) of tissue to saline was sufficient; after homogenization and lysis, centrifuge at 10,000 g for 10-15 min at 4°C, take the supernatant and the protein removal reagent 1:1 and mix thoroughly; centrifuge at 10,000 g for 10-15 min at 4°C, and remove the supernatant for subsequent reduction of glutathione assay;

1.1.3. Cell samples: After cell collection, cells can be resuspended using a suitable buffer, approximately 300-500 μL of PBS or saline per approximately every 1 X 106 cells; after homogenization, take the supernatant and the protein removal reagent 1:1 and mix thoroughly; centrifugation is performed at 4°C, 10,000 g for 10-15 min, and the supernatant is extracted for the subsequent reduced glutathione assay;

1.2. Sample pre-treatment: Glutathione standard (GSH powder) was centrifuged on a low-speed centrifuge to ensure that the powder was at the bottom of the tube; it was dissolved in 500 μL of sterile ultrapure water to formulate reduced glutathione standard (1 mmol/L), and it is recommended to store it at -20 after dispensing and use it up within 1 month.

1.3. Standard preparation: Reduced glutathione standard (1 mmol/L) was gradient diluted using ultrapure water (e.g., 50 μL of its standard was added to 950 μL of ultrapure water, diluted to 50 μmol/L and further gradient diluted to make a standard curve, i.e., 50/25/12.5/6.25/3.125 μmol/L), and then used for data analysis by the standard curve method after subsequent testing with the samples to be tested.

2. Reduced glutathione assay:

2.1. Mix the assay probe and ultrapure water 1:19 to form the assay probe working solution (recommended for ready-to-use);

2.2. Refer to the table below for reduced glutathione assays;

3. Data analysis:

3.1. Standard curve method:

3.1.1. Plot the standard curve according to the gradient dilution of the standard group value - blank group value data using Excel and other software: Y = a X + b ;

3.1.2. Sample reduced glutathione content calculation:

GSH content per unit serum sample (μmol/L) = (ΔA-b) ÷ a × 5^* × f 

GSH content per unit tissue sample (μmol/gprot) = (ΔA-b) ÷ a × 2^* × f ÷ protein concentration 

3.1.3. GSH content per unit cell sample (μmol/gprot) = (ΔA-b) ÷ a × 2^* × f ÷ protein concentration

Y is the standard group value - blank group value; X is the reduced glutathione content; a is the slope of the standard curve; b is the standard curve intercept; ΔA is: sample group value - blank group value; 5^* and 2^* are the number of times serum and protein removal reagents were diluted after mixing;; f is the number of times diluted.

3.2. Standard product conversion method:

3.2.1. Sample reduced glutathione content calculation:

Unit serum GSH content (umol/L) = ΔA/ΔA1 × standard tube concentration × 5^*

GSH content per unit of tissue (μmol/gprot) = ΔA/ΔA1 × standard tube concentration × 2^* ÷ protein concentration

GSH content per unit cell sample (μmol/gprot ) = ΔA/ΔA1 × standard tube concentration × 2^* ÷ protein concentration

ΔA is the absorbance value of the sample group - absorbance value of the blank group; ΔA1 is the absorbance value of the standard tube - absorbance value of the blank group; the concentration of the standard tube (300 μmol/l); and 5^* and 2^* is the multiplicity of the dilution of the serum and the protein removing reagents after mixing.

Note

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

2. The first use can be properly packaged and stored according to the situation to avoid repeated freezing and thawing affecting the performance of the reagent.

3. The Protein Removal Reagent will affect the BCA assay of the sample, please perform a BCA assay on homogenized samples prior to adding this component.

4. When the standard product conversion method analyzes the data, the concentration of the sample to be tested and the standard product must be within the linear detection range of the kit. When analyzing data with the standard curve method, ensure that the standard curve R2 is greater than 0.99.

5. Reducing agents and sulfhydryl active compounds can seriously 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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