Patented detection of GSH and GSSG in one well
INTRODUCTION
The DetectX® Glutathione kit is designed to quantitatively measure
GSH and GSSG present in a variety of samples. Total glutathione is the sum
of the oxidized form plus the GSH. A GSH standard is provided to generate a
standard curve. The kit utilizes a proprietary non-fluorescent molecule,
ThioStar®, that will covalently bind to the free thiol group on GSH to yield a
highly fluorescent product. After mixing the sample or standard with ThioStar®
and incubating at room temperature for 15 minutes, the GSH-generated signal
is read at 510 nm in a fluorescent plate reader with excitation at 390 nm.
Addition of a reaction mixture that converts all the GSSG into free GSH which
then reacts with the excess ThioStar® during a second 15 minute incubation
yields the signal related to Total GSH content.
Glutathione (GSH) is the highest concentration non-protein thiol in mammalian cells and is present in
concentrations of 0.5 to 10 mM. GSH plays a key role in many biological processes, including the synthesis of
proteins and DNA, the transport of amino acids, and the protection of cells against oxidation. Harmful hydro-
gen peroxide cellular levels are minimized by the enzyme glutathione peroxidase (GP) using GSH as a
reductant. The oxidized GSH dimer, GSSG, is formed from GSH and peroxide by the GP reaction. An
important role of GSSG in the NFkB activating signal cascade is suggested by the fact that the potent NFkB
inducer TPA increases intracellular GSSG levels and GSSG/GSH ratios.
SAMPLE TYPES
Blood, serum, plasma, urine, erythrocytes and cell lysate samples have been validated with this assay.
TYPICAL DATA
info@ArborAssays.com • www.ArborAssays.com
One sample measurement of GSH and GSSG
Fluorescent assay perfect for HTS
1 Plate
5 Plate
FEATURES
Catalog Number K006-F1
Catalog Number K006-F5
DetectX®
Glutathione Fluorescent Detection Kit
1.66
1.59
1.75
1.68
105.6%
105.7%
0.87
0.83
0.89
0.86
102.4%
104.1%
Mean Recoveries
101.5%
102.9%
24 Jurkat cell lysate samples were run in this kit and one based on Ellman’s colorimetric read-
out according to the manufacturers directions.
The slope and correlation coefficient were 0.923 and
0.991 respectively.
Four urine samples gave Total GSH readings of between 0.63 and 4.04 µM in
the kit. In the Ellman’s based kit none of the urine samples could be detected due to lack of sensitivity.
For details concerning this kit or to order any of our products please contact us:
734-677-1774 • info@ArborAssays.com • www.ArborAssays.com
3
9.38
11.67
4
4.89
5.89
KIT CORRELATION DATA
0.269
0.564
0.354
3.57
2.40
0.112
1
1.30
2
1.83
Total Std. Dev.
( M)
0.198
0.356
0.699
0.474
Free %CV
Total %CV
8.6
8.3
14.7
10.0
6.0
6.0
7.2
8.0
Sample
Free GSH Conc. Total GSH Conc. Free Std. Dev.
( M)
( M)
( M)
INTER ASSAY PRECISION
Two each of SSA treated human urine and whole blood samples were run in duplicates in multiple assays
over multiple days by two operators. The mean and standard deviation of the calculated GSH concentrations
were:
SAMPLE PREPARATION
All samples need to be deproteinized prior to use. Detailed protocols for all samples are included in the kit insert.
ASSAY PROTOCOL
• Pipet 50 L of samples or standards into duplicate wells in the microtiter plate.
• Add 25 L of the ThioStar® Reagent to each well.
• Incubate at room temperature for 15 minutes. Read free GSH in a fluorescent plate reader at 510 nm
(Exc=390nm).
• Add 25 L of Reaction Mixture to each well. Incubate at room temperature for 15 minutes.
• Read Total GSH in a fluorescent plate reader at 510 nm (Exc=390nm).
SENSITIVITY
Sensitivity was determined as < 50 nM GSH.
LINEARITY
Linearity was determined by taking 2 Jurkat cell lysate samples one, with 1.28 x 106 cells/mL and one with a
higher level of 25 x 106 cells/mL, and mixing them in the ratios given below:
High Cell # Low Cell #
Free Exp.
Total Exp.
Free Obs.
Total Obs. % Recovery % Recovery
%
%
Conc. ( M)
Conc. ( M) Conc. ( M) Conc. ( M)
Free
Total
80%
20%
3.24
3.11
3.15
3.09
97.3%
99.5%
60%
40%
2.45
2.35
2.47
2.41
100.8%
102.5%
0.107
4.0
4.7
0.178
3.1
4.7
0.261
4.6
2.7
0.101
3.0
2.3
40%
60%
20%
80%
INTRA ASSAY PRECISION
Two each of SSA treated human urine and whole blood samples were run in replicates of n=20. The mean
and standard deviation of the calculated GSH concentrations were:
Sample
Free GSH Conc. Total GSH Conc. Free Std. Dev. Total Std. Dev.
Free %CV
Total %CV
( M)
( M)
( M)
( M)
1
1.27
2
2.00
3
8.33
4
3.89
2.30
0.051
3.80
0.063
9.77
0.381
4.45
0.115
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ