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Glutathione ELISA as...

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Glutathione ELISA assay complete guide


About:
glutathione (r-glutamyl cysteingl +glycine (GSH)) is a tripeptide containing gamma-amide bonds and thiol groups, composed of glutamic acid, cysteine, and glycine, which is present in almost every cell of the body. Glutathione helps maintain normal immune system function and has antioxidant, integrative and detoxifying effects. The sulfhydryl group on cysteine is its active group (often abbreviated as G-SH), which is easy to combine with some drugs, toxins, etc., so that it has an integrated detoxification effect.

Operation steps:

First, material preparation

1. Coated liquid: 0.05mol /l (pH9.6) carbonate buffer: Na2CO3 0.159g, NaHCO3 0.294g, dissolved with distilled water, and the volume was fixed to 100 ml.

2. PBS-T washing liquid: 0.01mol/ pH7.4 phosphate-sodium chloride buffer: NaCl 8.0g, KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, KCl 0.2g, Tween-20 0.5ml, dissolved in distilled water, constant volume to 1000 ml.

3. Substrate solution 3, 3 ', 5,5 '-tetramethylbenzidine (TMB) storage solution and application solution:
(1) 0.1mol /l pH6.8 PBS: Na2HPO4·12H2O 1.09 g, NaH2PO4·H2O 6.05 g, distilled water dissolved to 500 ml, stored at 4℃ for later use.
(2) TMB storage solution: TMB 60 mg dissolved in 10 ml dimethyl sulfoxide (DMSO) at 4℃ for storage.
(3) TMB application solution: Before use, take 0.1 mol/l pH6.0 PBS 10 ml, TMB reserve solution 100 ul, 30%H2O2 15 ul and mix well.

2. Experimental steps

1. Antibody coating
(1) The antibody was diluted to 20 ug/ml with coated solution, added to the polystyrene reaction plate with 100 ul per well, covered and left at 4℃ overnight.
(2) Pour out the liquid in the hole, add the washing liquid and let it stand for 3 minutes, drain the washing liquid, and repeat washing for 3 times.
(3) Place the mouth of the reaction plate on the absorbent paper to remove the liquid.

2. Sealing: Add 200 ul sealing solution to each well, incubate at 37℃ for 60 min, and then wash 3 times.

3. Add the serum to be tested, incubate at 37℃ for 1~2 h, and wash 3 times. The standard curve was prepared by using diluent pure GST-π.

4. Add enzyme-labeled antibody: Dilute anti-GST-π-IGg-HRP according to requirements, 100 ul per well, incubate at 37℃ for 1 h, then wash 5 times, and blot on absorbent paper.

5. Color rendering: Add TMB application solution, reaction at room temperature for 15~30 min, add 50 ul 2 mol/l H2SO4, stabilize for 3~5 min, you can compare colors.

6. Colorimetry: Using enzyme marker, with PBS-T hole as the control, wavelength 450 nm, measure the light absorption (A) of each hole, check the standard curve, you can measure the GST-π content.

References:
1. [Title] Tissue engineered artificial liver model based on viscoelastic hyaluronan-collagen hydrogel and the effect of Tissue Engineered Artificial Liver Model EGCG intervention on ALD
2. [Title] Analysis of the effect of probucol-mecobalamin tablets combination on oxidative stress in patients with diabetic patients  peripheral neuropathy
3. [Title] Effects of sublethal concentrations of cyantraniliprole on the biology and metabolic enzyme activities of the Body Laodelphax striatellus (Fall′en)
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