Total Glutathione/Oxidized Glutathione 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. Moreover, this kit provides GSH scavenger, which can be used to remove the original GSH in the sample, and the remaining GSSG is converted to GSH again under the action of glutathione reductase, and then reacted with DTNB, so as to measure the GSSG content.

Storage and Shipping Conditions

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

Product Content

Note: If simultaneous detection of T-GSH and GSSG is required, the number of kit assays is half of the corresponding specification.

Assay Protocol / Procedures

1. Sample Preparation:

1.1. Sample pre-treatment

1.1.1. Plasma and serum samples: add equal volume of protein removal reagent, thoroughly mix; Centrifuge at 10,000g at 4 for 10-15 min and take the supernatant for subsequent detection;

1.1.2. Tissue samples: add protein removal reagent to the tissue for homogenization and lysis (Weight mg: volume μL is 1:9, e.g., 20 mg to 180 μL); after homogenization and lysis, centrifuge the tissue at 10,000 g for 10-15 min at 4°C, and remove the supernatant for subsequent detection;

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, and lysed by sonication or on ice for 10-20 min; after treatment, centrifuge at 4°C, 10,000 g for 10-15 min, and remove the supernatant for subsequent assays;

1.2. Standard preparation:

1.3. Glutathione standard (GSSG powder) was centrifuged on a low-speed centrifuge to ensure that all the powder was at the bottom of the tube; it was dissolved using 500 μL of sterile ultrapure water to prepare glutathione standard (GSSG, 1 mmol/L), and it is recommended to dispense it and store it at -20°C, and use it up within 1 month.

1.4. protein removal reagent and ultrapure water are mixed 1:9 and used to make a gradient dilution (For example, 40 μL of the standard was added to the above 960 μL mixture, diluted to 40 μmol/L, and further gradient dilution to make a standard curve, i.e., 40/20/10/5/2.5 μmol/L) of glutathione standard (1 mmol/L), which is used for data analysis by the standard curve method after subsequent detection with the samples to be tested.

1.5. Configure Glutathione Assay Buffer: Glutathione Reductase: Assay Probe = 150:10:1 to form Glutathione Assay Working Solution;

2. Total glutathione (T-GSH) assay:

2.1. The total glutathione test sample is processed as follows (Note: Because the GSSG assay requires sample pre-treatment, to ensure system consistency, samples should be pre-treated with the same system before performing the total glutathione assay);

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

3. Oxidized glutathione (GSSG) assay:

3.1. Refer to the table below for clearance processing of reduced glutathione;

3.2. Refer to the following table for assay of oxidized glutathione (The sample to be tested here is the sample treated in step 3.1)

4. Data analysis:

4.1. Standard curve method:

4.2. T-GSH and GSSG standard curves were plotted using Excel and other software based on the gradient dilution standard group value-blank group value data: Y1=a1 X1+ b1 and: Y2=a2 X2+ b2 ;

4.2.1. Calculation of T-GSH and GSSG content in blood samples:

T-GSH content per unit serum sample (μmol/L) = (ΔA1-b1) ÷ a1 × 2^* × 5^* × f 

4.2.2. GSSG content per unit serum sample (μmol/L) = (ΔA2-b2) ÷ a2 × 5^* × f

4.3. Calculation of T-GSH and GSSG content in tissue samples:

T-GSH content per unit tissue sample (μmol/kg) = (ΔA1-b1) ÷ a1 × V ÷ m × 2^* × f 

4.4. GSSG content per unit tissue sample (μmol/kg) = (ΔA2-b2) ÷ a2 × V ÷ m × f

4.5. Calculation of T-GSH and GSSG content of cell samples:

T-GSH content per unit cell sample (μmol/10^9cell) = (ΔA1-b1) ÷ a1 × V ÷ n × 2^* × f 

GSSG content per unit cell sample (μmol/10^9cell) = (ΔA2-b2) ÷ a2 × V ÷ n × f

Note: Reduced glutathione content (μmol/L) = Total glutathione content (μmol/L) - 2 x oxidized glutathione (μmol/L)

Note: Y1 and Y2 are OD values of T-GSH and GSSG standard group-OD value of blank group, respectively; X1 and X2 are T-GSH and GSSG contents, respectively; a1 and a2 are slopes of T-GSH and GSSG standard curves, respectively; b1 and b2 are intercepts of T-GSH and GSSG standard curves, respectively; ΔA1 and ΔA2 are T-GSH and GSSG: OD value of sample group-OD value of blank group, respectively; 5* is the multiple of dilution after mixing serum and protein removal reagents; 2^* is the multiplication of 2 when converting GSSG to GSH; V is the value of homogenization OD group. OD value-blank OD group value; 5^* is the number of times serum and protein removal reagent are mixed and diluted; 2^* is the number of times GSSG needs to be multiplied by 2 when converted to GSH; V is the volume of protein removal reagent used for homogenization of lysed tissues or cells (mL); m is the wet weight of tissues (g); n is the amount of cells (106); and f is the number of times diluted.

Note

1. Fully dissolve and gently shake the various reagents before use to ensure that the components are used under homogeneous conditions.

2. The content of different samples varies, so please increase or decrease the concentration of the samples appropriately according to the actual situation.

3. This kit can detect only total glutathione or oxidized glutathione, or both, as needed.

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

5. When the final calculation is abnormal, it may be that the sample GSH scavenging is abnormal and the amount of GSH scavenger used or the incubation time can be increased or decreased as appropriate.

6. 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.

7. When analyzing data using the standard curve method, ensure that the standard curve R2 is > 0.99.

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

9. GSH Remover has a pungent odor, and should be handled in a fume hood.

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

Appendix: Technical parameters

For Research Use Only!

https://www.servicebio.com/goodsdetail?id=60803