Glutathione (glutathiose, r-glutamyl cysteingl + glycine, GSH) is a tripeptide containing a γ-amide bond and a thiol group, which is composed of glutamic acid, cysteine ​​and glycine. It exists in almost every cell of the body.

Experimental method Double-anti-sandwich ELISA immunohistochemical method The principle of the method is to first coat the body on the reaction plate, add the antigen to be tested, and then add the enzyme standard body, and the antigen content can be measured after color development.
Experimental material antibody
Reagents, kits carbonate buffer Na2CO3 phosphate buffer NaCl KCl TMB dimethyl sulfoxide bovine serum albumin
Instrument, consumables, microplate incubator
Experimental procedure 1. Material preparation

1. Coating solution: 0.05 mol/l (pH 9.6) carbonate buffer: Na2CO3 0.159 g, NaHCO3 0.294 g, dissolved in distilled water, and made up to 100 ml.

2. PBS-T washing solution: 0.01mol/pH 7.4 phosphate-sodium chloride buffer: NaCl 8.0 g, KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, KCl 0.2 g, Tween-20 0.5 ml, with distilled water After dissolution, dilute to 1000 ml.

3. Substrate solution 3,3',5,5'-tetramethylbenzidine (TMB) stock solution and application solution:

(1) 0.1 mol/l pH6.8 PBS: Na2HPO4·12H2O 1.09 g, NaH2PO4·H2O 6.05 g, dissolved in distilled water to a volume of 500 ml, and stored at 4 ° C for use.

(2) TMB stock solution: TMB 60 mg is dissolved in 10 ml of dimethyl sulfoxide (DMSO) and stored at 4 ° C for use.

(3) TMB application solution: Before use, take 0.1 mol/l pH6.0 PBS 10 ml, TMB stock solution 100 ul, 30% H2O2 15 ul and mix well.

Second, the experimental steps

Antibody coating

(1) The antibody was diluted to 20 ug/ml with a coating solution, and added to 100 ul per well in a polystyrene reaction plate, and placed at 4 ° C overnight.

(2) The liquid in the well was decanted, and the washing liquid was added and allowed to stand for 3 min, and the washing liquid was drained, and the washing was repeated 3 times.

(3) The reaction plate is placed on the absorbent paper to remove the liquid.

2. Blocking: Add 200 ul of blocking solution to each well, incubate at 37 °C for 60 min, then wash 3 times.

3. Add the serum to be tested, incubate at 37 °C for 1~2 h, and wash 3 times. The dilution was used as a control well and the standard curve was prepared using pure GST-π which is not tolerant to dilution.

4. Add enzyme-labeled antibody: Dilute anti-GST-π-IgG-HRP as required, 100 ul per well, incubate at 37 °C for 1 h, then wash 5 times, and buckle on absorbent paper to absorb water.

5. Color development: Add TMB application solution, set room temperature reaction for 15~30 min, add 50 ul 2 mol/l H2SO4, stabilize for 3~5 min, then colorimetric.

6. Colorimetric: GST-Ï€ content can be measured by using a microplate reader with PBS-T well as control, wavelength 450 nm, measuring the light absorbance (A) of each well, and checking the standard curve.

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