Reduced GSH oxidizes rapidly, what should I do to aviod oxidization during the biochemical analysis of tissue sample? I found some method to measure it are based on Ellman reaction, however this reaction is for all available thiol group, how to explain it?
Thanks in advance
Dear Li,
I analysed GSH concentration in amphipod tissue during my PhD thesis, by using HPLC method. Article Influence of gender and season on reduced glutathione concen...
To homogenate my tissue, I used a buffer containing a protease inhibitors mixture, and after centrifugation, the supernatant was added with perchloric acid to eliminate all proteins from the sample.
Ellman reaction is based on a specific reaction with thiol function (-SH), so it's normal to have all thiol group measured together.
There are also some GSH kit... See Cayman supplier.
I hope it will help you.
Good luck
Eric
Please find the attached paper for a simple method of thiol/disulfide measurement.
Hi Li,
Glutathione oxidizes rapidly and is a suitable substrate for γ-glutamyl transpeptidase. Therefore, it should be acidified quickly to avoid oxidation and inactivate γ-glutamyl transpeptidase when you assay tissue samples. GSH tends to oxidize faster when the pH goes beyond 7.
Assay the samples asap after the dissection; organs rinsed in DW>blotted dry, and;
Larger tissue can be homogenized in 5 (vol/g (wet tissue)) in 5% 5-sulfosalicylic acid.
Smaller tissues can be homogenized in 10 (vol/g (wet tissue)) in 5% 5 sulfosalicylic acid.
Homogenate should be centrifuged at 10,000g for 5 min, then store the supernatant at 4C until assayed later.
See this ref:
Anderson et al, 1985. Determination of Glutathione and Glutathione Disulfide in Biological Samples
Commercial kit: https://www.cellbiolabs.com/total-thiol-assay
I hope this helps.
Charith
Read the following articles:
https://www.researchgate.net/publication/274956472_A_practical_HPLC_method_to_measure_reducedGSH_and_oxidized_GSSG_glutathione_concentrations_in_animal_tissues
https://pubmed.ncbi.nlm.nih.gov/12815674/
Use DTNB method. See the attached PDF for details. All the best.
Prof. Katyare
Hi, Ellman sometimes used as a measure of total thiols as the regent DTNB reacts with a thiol group, bur, using TCA in the procedure or precipitating all other thiol protiens will make the measurement more accurate.
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