Application

This kit can be used to measure the GSH content in animal serum, plasma, tissue, whole blood, culture cells and other samples.

Detection significance

Reduced Glutathione (GSH) is a kind of low molecular scavenger, which can remove O2-, H2O2, LOOH. GSH is a small molecule peptide which composed of glutamic acid, glycine and cysteine, and it is the main thiol compound of non-protein in the organization. GSH is the substrate of GSH-PX and GSH-ST which is indispensable for decomposing hydrogen peroxide of these two enzymes, and it can stabilize the enzyme containing thiol and prevent hemoglobin and other auxiliary factors from the oxidative damage. Recently, it is proved that GSH is also involved in the recovery of VE to the reduction state. When lacking or depletion of GSH, it may cause producing toxic effects or increasing the toxic effects of many chemicals or environmental factors. It may be related to the increase of oxidative damage, so the amount of GSH is a vital factor to measure the body's antioxidant ability. GSH plays an important role in the researches process of prevention, recovery and treatment of atherosclerosis, coronary heart disease, anti-aging, anti-tumor, prevention, prevention of Alzheimer's disease and other diseases.

Detection principle

Reduced GSH can react with Dinitrobenzoic acid (DNTB) to form a yellow complex which can be detected by colorimetric assay at 405 nm and calculate the reduced GSH content indirectly.

Experimental instrument

Microplate (96 wells), Micropipettor, Vortex mixer, Centrifuge, Microplate reader (405 nm)

Sample pretreatment

1. Culture cells:

Collecte the cells and wash 1~2 times with PBS. Centrifuge with low speed and collect the cells. Add 0.3~0.5 mL of PBS buffer (0.1 M, pH 7.4) to resuspend the cells. Sonicate or grind with hand-operated in ice water bath to break the cells.

Preparation of supernatant: take 0.1 mL cell sample, add 0.1 mL of Reagent 1 and mix fully. Centrifuge at 4500 g for 10 min. Take the supernatant for detection.

2. Tissue samples:

Preparation of 10% tissue homogenate: Accurately weigh the tissue, add normal saline at a ratio of Weight (g): Volume (mL) =1:9 and homogenize the sample in ice water bath. Then centrifuge at 2500 rpm for 10 min, then take the supernatant for detection.

Preparation of supernatant: take 0.1 mL 10% tissue homogenate, add 0.1 mL of Reagent 1 and mix fully. Centrifuge at 4500 g for 10 min. Take the supernatant for detection.

3. Serum (plasma):

Take 0.1 mL of serum (plasma), add 0.1 mL of Reagent 1 into and mix fully. Centrifuge at 4500 g for 10 min. Take the supernatant for detection.

4. Whole blood sample:

Take 0.1 mL of the whole blood anticoagulated with heparin, add 0.1 mL of Reagent 1 and mix fully. Centrifuge at 4500 g for 10 min. Take the supernatant for detection.

Operation steps

1. Add 25 μL Reagent 3 to each tube.

2. Blank tube: Add 100 μL of Reagent 1.

Standard tube: Add 100 μL of 40 μmol/L GSH standard solution.

Sample tube: Add 100 μL of supernatant.

3. Add 100 μL of Reagent 2 to each tube.

4. Mix fully for 1 min and stand for 5 min. Measure the OD values of each well at 405 nm with Microplate reader.

Note: The following operating table could be as a reference.

Technical parameter

1. The sensitivity of the kit is 2 μmol/L.

2. The intra-assay CV is 1.9% and the inter-assay CV is 3.2%.

3. The recovery of the kit is 96%.

4. The detection range of the kit is 2-400 μmol/L.

Notes

1. This kit is for research use only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

4. Do not use components from different batches of kit.