Measurement of Oxidized/Reduced Glutathione Ratio - Protein Misfolding and Cellular Stress in Disease and Aging - page 263

8. Add 150 mL of assay cocktail to each well, cover plate to
ensure complete darkness, and incubate the plate on an orbital
shaker to ensure thorough mixing.
9. Insert plate into plate reader and start measurements (see
Note 5).
10. Measure the concentration of GSH using either the end-
point or kinetic method. Use the kinetic method if levels
of cysteine and other free thiols are expected to be high
(see Note 6).
11. The endpoint method requires the average absorbance value
(do not forget to subtract the blank) of each standard at the
25 min interval to be plotted vs. standard concentration. Use
the following equation to calculate GSH concentrations:

{(Absorbance at 414 nm) − ( Y - intercept)}

[Total GSH] = ×× 2 Sample Dilution

i-slope

12. The kinetic method plots the resultant slope of each standard
( i (i-slope) vs. GSH concentration. Take measurements every
5 min for 30 min to obtain the i i-slope values (see Note 5).
The standard slopes are plotted against the standard GSH
concentrations and the plot slope ( f -slope) is used in the
equation below to calculate the sample GSH concentrations:

{(Sample - slope) i − ( Y - intercept)}

[Total GSH] = ×× 2 Sample Dilution

f - slope

The i i-slope value for each sample is calculated and inserted
into the equation above (see Note 7).

3.5. DTNB-GSSG
Enzymatic Recycling
Assay for GSSG

1. Follow steps 1-4 in Subheading 3.4 Sample Deproteination.
2. Add 10 mL of 2-vinylpyridine solutions for every mL of sample.
3. Vortex sample and allow the solution to sit at room tempera-
ture for 1 h.
4. This procedure has a detection limit of 1 mM GSSG. If sam-
ples are suspected of concentrations greater than 1 mM, add
TEAM to dilute sample.
5. Treat all standards as described in steps 1-4. 2-Vinylpyridine
inhibits color development, and it is important that all sam-
ples and standards are incubated for the same amount of time
and with identical 2-vinylpyridine treatments.
6. Follow steps 1-12 outlined in Subheading 3.4 'DTNB-GSSG
Enzymatic Recycling Assay for Total GSH' to measure total
GSSG level.