I homogenize my sample with PBS and then I add solphosalcylic acid %4 in same mount in the supernatant. The sample incubated for 30min in 4c and then centrifuged in 4000g for 10min and I add prepared Ellman reagent the samples and incubated for 15 min in room temperature but i dont see yellow color. I am sure my Ellman reagent is active and I think that my sample preparation has problem. Does anybody have any experience in GSH measurement to help me??
Dear Parisa Faraji
I conducted a comprehensive study, in part it can help (I think we need to collect a fraction and it can be converted into a quantitative volume using statistical programs).
Blood was quickly removed from the brain, separated from the meninges and the studied pieces were placed in liquid nitrogen. Then, they were ground in liquid nitrogen to a powder and homogenized in a 10-fold volume of the medium at (2°С) containing (in mmol): sucrose, 250; TrisHCl buffer, 20; EDTA-1 (pH 7.4). At a temperature (+4°C) by the method of differential centrifugation on a refrigerated centrifuge Sigma 3-30k separated the cytosolic and mitochondrial fractions (17000 g).
The supernatant was discarded and stored at –80°С. The state of the thiol-disulfide system of the brain was assessed by the content of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the cytosolic fraction fluorimetrically using o-phthalic anhydride on a Quantech fluorometer. Level of free
SH-groups, activity of glutathione reductase, glutathione peroxidase and glutathione transferase in
the cytosolic fraction was measured spectrophotometrically on a Libra S 32 PC spectrophotometer. The content of cysteine and methionine was determined by thin layer chromatography in the cytosolic fraction followed by spectrophotometry on a spectrophotometer. The total content of reduced and oxidized sulfhydryl groups in the cytosolic fraction was determined using Elman's reagent spectrophotometrically on a spectrophotometer. The protein concentration was assessed by the Bradford method.
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