GSH + GSSG
(Tietze) Assay
Contact: Pietro, Brie, Ometa Herman
|
Materials |
Qty |
Location |
Order Info |
|
Na phosphate buffer |
|
Over Pietro’s bench, B-010 |
0.125M, pH 7.5 Naphosphate + 6.3mM tetrasodium-EDTA |
|
Na phosphate buffer+ 5% sulphosalicyclic acid |
|
|
Prepare fresh |
|
NADPH |
|
B-011 freezer Tietze dessicator |
Sigma N-1630 |
|
DTNB |
|
Over Pietro’s bench, B-010 |
Sigma D-8130, 5,5’-Dithio-bis(2-nitrobenzoic acid) |
|
GSH |
|
B-011 freezer Tietze dessicator |
Sigma G-4251 |
|
GSH reductase |
|
B-011 freezer Tietze dessicator |
Sigma G-3664 |
Reference: Methods of Enzymatic Analysis. HV Bergmeyer. 6/10/98
Prepare reagents
1) NADPH. Make up a 0.3mM solution (0.25mg/mL) in Na phosphate buffer
2)
DTNB. Make up a 6mM solution (2.378mg/mL) in Na
Phosphate buffer
3)
GSH. Make up a 1mg/mL solution (Note: will be used 1:60
later)
4)
GSH reductase (Boehringer). Prepare as needed for
20uL/test at 0.6U/20uL (Current vial is 173U/mg, meaning 1uL=1.38U)
Assay
1) Combine 100uL DTNB solution (at 2.378mg/ml) and 700uL NADPH solution (at 0.25mg/mL) in a cuvette for each test (make plenty for standards, samples, and blanks).
2)
Dilute GSH solution (1mg/mL) 1:60 in Na Phosphate
Buffer
3)
Warm up spectrophotometer. Set at Kinetic reading
(Method) at 412nm wavelength. Set for 5 second reading intervals, 0 sec
incremental time, and 6 readings/minute.
4)
Measure first two blanks with the spectrophotometer
(with nothing but NADPH and DTNB in buffer), adding 20uL GSH reductase to each
1 minute before reading.
5)
Measure standard samples to generate a standard ratio.
Read multiple standard dilutions of GSH from 5uL-150uL to identify which
concentrations will generate a linear curve of absorbance over time. Use the
table below for standard concentrations.
|
uL GSH |
ug/mL GSH |
|
5 |
0.083 |
|
10 |
0.176 |
|
25 |
0.415 |
|
50 |
0.83 |
|
75 |
1.25 |
|
100 |
1.67 |
|
150 |
2.5 |
Add the GSH to the cuvette (with 800 uL DTNB and NADPH from step 1). Let incubate at least 2 minutes. Add cuvette to sample holder in spectrophotometer (using DTNB+NADPH+GSH reductase from Step 4 as reference). Immediately before reading cuvette, add 20uL GSH reductase solution (after 1:60 dilution) and close door. Read immediately.
Stop at volume of GSH where the
curve for absorbance over time is no longer linear.
6)
Sample
measurements are expected to be within the range of dA/min measurements acquired
through the standards. To do this, dilutions of the sample may be necessary.
7)
Measure
samples. Add sample to cuvette (with DTNB and NADPH as above), let incubate for
at least 2 minutes. Place cuvette in spectrophotometer, then add 20uL