Measurement of glutathione
Glutathione (GSH) is a ubiquitous tripeptide which serves several vital functions including detoxifying electrophiles, scavenging free radicals and providing a reservoir for cysteine. The sulfhydryl group of GSH reduces 5-5'-dithiobis (2-nitrobenzoic acid) (DTNB) and form the yellow colour product 5-thio-2-nitrobenzoic acid (TNB)21. Measurement of the absorbance of TNB at 412 nm provides an accurate estimation of GSH in the sample. Figure 7.
Materials
5×105 cells/mL cell suspension
Culture medium, BSA, PBS (Ca2+, Mg2+ free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and amphotericin-B)
ENPs, Diethyl maleate (DEM)
5, 5'-dithiobis- 2-nitrobenzoic acid (DTNB), …show more content…
Methods
GSH estimation
• Take 500 µL of lysate supernatant and add 2.5 mL of DTNB solution.
• Vortex the solution and incubate at room temperature for 20 min.
• Dispense 200 µL of the solution in a 96 well (transparent bottom) plate in triplicate.
• Read on a microtiter plate at 412 nm.
Estimation of protein by Bradford assay
• Prepare the protein standard curve using the 1 mg/mL stock of BSA.
• Add 10 µL of known concentration of the BSA (ranging from 0.001 to 100 µg/mL) to 990 µL of Bradford reagent.
• Mix the solution well and incubate for 15 min.
• Add 10 µL of the test sample to 990 µL of Bradford reagent.
• Mix the solution well and incubate for 15 min.
• Dispense 200 µL of the sample reagent mixture to a 96 well (transparent bottom) plate in triplicate.
• Read on a microtiter plate at 595 nm.
• Calculate the concentration using the BSA standard curve.
GSH level in the test samples can be calculated by comparing the readings with GSH standard reading and expressed in equivalent GSH in μmoles/mg protein after normalization with total protein.
Limitations
• Cysteine, β-mercaptoethanol or dithiothreitol and other thiol containing groups may compete with GSH for
Glutathione (GSH) is a ubiquitous tripeptide which serves several vital functions including detoxifying electrophiles, scavenging free radicals and providing a reservoir for cysteine. The sulfhydryl group of GSH reduces 5-5'-dithiobis (2-nitrobenzoic acid) (DTNB) and form the yellow colour product 5-thio-2-nitrobenzoic acid (TNB)21. Measurement of the absorbance of TNB at 412 nm provides an accurate estimation of GSH in the sample. Figure 7.
Materials
5×105 cells/mL cell suspension
Culture medium, BSA, PBS (Ca2+, Mg2+ free), FBS, trypsin-EDTA, L-glutamine, antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and amphotericin-B)
ENPs, Diethyl maleate (DEM)
5, 5'-dithiobis- 2-nitrobenzoic acid (DTNB), …show more content…
Methods
GSH estimation
• Take 500 µL of lysate supernatant and add 2.5 mL of DTNB solution.
• Vortex the solution and incubate at room temperature for 20 min.
• Dispense 200 µL of the solution in a 96 well (transparent bottom) plate in triplicate.
• Read on a microtiter plate at 412 nm.
Estimation of protein by Bradford assay
• Prepare the protein standard curve using the 1 mg/mL stock of BSA.
• Add 10 µL of known concentration of the BSA (ranging from 0.001 to 100 µg/mL) to 990 µL of Bradford reagent.
• Mix the solution well and incubate for 15 min.
• Add 10 µL of the test sample to 990 µL of Bradford reagent.
• Mix the solution well and incubate for 15 min.
• Dispense 200 µL of the sample reagent mixture to a 96 well (transparent bottom) plate in triplicate.
• Read on a microtiter plate at 595 nm.
• Calculate the concentration using the BSA standard curve.
GSH level in the test samples can be calculated by comparing the readings with GSH standard reading and expressed in equivalent GSH in μmoles/mg protein after normalization with total protein.
Limitations
• Cysteine, β-mercaptoethanol or dithiothreitol and other thiol containing groups may compete with GSH for