Premier Products for Superior Life Science Research

NWLSSTM
Glutathione Assay
Product NWK-GSH01
For Research Use Only

5131 NE 94th Avenue, Suite 201
Vancouver, WA 98662
Phone 360-449-3091 or Toll Free: 888-449-3091
Fax 360-449-3092
E-mail: nwsales@nwlifescience.com
nwkgsh01rev010307

Simple assay kit for quantitative measurement ofglutathione in biological samples such as whole
blood, tissue homogenates and cell lysates.
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Page

Introduction

3

Intended Use

3

Test Principle

3

General Specifications

4

Kit Contents

4

Required Materials Not Provided

4

Required Instrumentation

4

Warnings, Limitations, Precautions

5

Storage Instructions

5

Assay Preparation

5

Reagent Preparation

6

Sample Handling/Preparation

6

Assay Protocol

8

Data Analysis

8

Performance Details

11

Things To Note

12References

13

Statement of Limited Warranty

13

Procedure Checklist

14

End-User Notes

15

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End-User Notes:

Table of Contents
SectionTo Order Call: 1-888-449-3091

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Procedure Checklist__
Process the sample and place on ice__

Prepare 5% MPA and place on ice__

Prepare Calibrators and place on ice__

Bring GSH reagents to ambient temperature__

Setup microplate reader
Wavelength = 405 nm
Mode = Kinetic
Reaction Time = 3 Minutes
Read Interval = 15-20 seconds (9-12 data points)__

Deproteinate samples 50:100; sample to MPA__Centrifuge sample for 1 minute at 10,000 x g__

Dilute samples 25:500; supernatant to Assay Buffer and place
on ice__Reconstitute NADPH Reagent with NADPH Reagent Diluent__

Add 50 µL calibrators, controls and diluted samples toappropriate wells of microplate__

Add 50 µL DTNB Reagent to all wells__

Add 50 µL GR Reagent to all wells__

Incubate 2-3 minutes at ambient temperature__

Add 50 µL NADPH Reagent__

Place microplate in plate reader and begin 405 nm
measurements__

Calculate results__

Return reagents to 2-8°C.

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Page 3Introduction:
Glutathione (GSH, γ -glutamylcysteinylglycine), the primary non-protein
sulfhydryl in aerobic organisms is synthesized in most cells. The ubiquitoustripeptide is formed by the ATP dependent condensation of glutamic acidand cysteine, catalyzed by γ-glutamylcysteinyl synthetase. Glycine is thenadded by glutathione synthetase to form GSH.
+

O
O
NH3
OH
C C CH2 CH2 C N CH C N CH2 C
H
H
H
O
O
CH2

O

SH

Figure 1.
Structure of GSH
In addition to donating an electron during the reduction of hydroperoxides
to the respective alcohols (or water in the case of hydrogen peroxide), GSH
functions as a co-substrate in the metabolism of xenobiotics catalyzed byglutathione S-transferases. It is also a co-factor for several metabolic
Enzymes, is involved in intracellular transport, functions as an antioxidant
and radioprotectant and facilitates protein folding and degradation.
Intended Use:
The NWLSS™ Glutathione Assay is used to measure the concentration of
total GSH (reduced and oxidized) in a variety of animal and plant samples.
Test Principle:
The NWLSS™ Glutathione Assay is a modification of the method firstdescribed by Tietze.2 The general thiol reagent, 5-5′-dithiobis[2-nitrobenzoicacid] (DTNB, Ellman’s Reagent) reacts with GSH to form the 412 nm
chromophore, 5-thionitrobenzoic acid (TNB) and GS-TNB. The GS-TNB is
subsequently reduced by glutathione reductase and β-nicotinamide
adenine dinucleotide phosphate (NADPH), releasing a second TMB molecule and recycling GSH; thus amplifying the response. Any oxidized GSH(GSSG) initially present in the reaction mixture or formed from the mixed
disulfide reaction of GSH with GS-TNB is rapidly reduced to GSH.
GSSG
GR
TNB

GSH

DTNB

GSTNB

TNB

GR

Figure 2.
GSH Recycling Pathway
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General Specifications:
Format::2 X 96 wells
Number of tests: Triplicate =Duplicate =58
90

Specificity:

Total Glutathione (GSH plus GSSG)

Sensitivity:

LLD = 0.1 µM in the reaction(Calculated as 3.29 standard deviations from zero).

Kit Contents:
NADPH:

β-Nicotinamide adenine dinucleotide phosphate,
reduced; 2 vials powder.

NADPH Diluent:

Buffer with stabilizer; 2 x 6 mL vials.

DTNB:

5-5′-Dithiobis(2-nitrobenzoic acid) in phosphate buffer
with EDTA; 1 x 11 mL.

GR Enzyme:

Glutathione reductase in Assay Buffer with protein
stabilizer; 1 x 11 mL.

Calibrators:

Glutathione disulfide in 1 mM HCl, 400 µM GSH
equivalents; 1 x 0.5 mL.

Assay Buffer:

Phosphate buffer with EDTA, pH 7.6; 1 x 125 mL

Required Materials Not Provided:
Pipettes capable of transferring 25, 50, 100 and 500 µL volumes.
A multi-channel or repeater pipette (recommended)
Metaphosphoric acid , (Sigma 239275 or equivalent).
Deionized water
Polypropylene microcentrifuge tubes or equivalent
Stir bar
Beaker or flask 25-50 mL
Required Instrumentation:
Microplate reader with kinetics capability at 405 nm.
Note: The λmax for TNB is 412 nm, however most plate readers
are equipped with a 405 nm filter.

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Things to Note: (continued):
4. NADPH that has been stored for a prolonged time can be tested bydemonstrating that the rate curve for the high calibrator is linear over a 5
minute interval.
5. Unused wells can be protected from contamination by used wells by
sealing the used wells with 1 inch cellophane tape.
6. Samples containing GSH concentrations greater than the upper limit of
the assay can usually be detected by observing very rapid color development. The rate curves of suspicious sample should be examined for linearity. Generally the reaction rate can be re-calculated over the linear portion of the rate curve and used to calculate the GSH concentration.
7. GSH is rapidly metabolized and will undergo mixed disulfide reactions;
therefore, it is recommended that samples be processed as soon as possible to avoid the loss of GSH.
8. Please contact NWLSS for advice if the expected GSH concentration in
the samplers is near or below the sensitivity of the standard method.
References:
1. Halliwell, B.; Gutteridge, J.M.C. Free Radicals in Biology and Medicine.
Oxford University Press, New York; 1999.
2. Teitze, F. Enzymatic method for the quantitative determination of
nanogram amounts of total and oxidized glutathione: Applications tomammalian blood and other tissues, Anal. Biochem.27:502-522; 1969.
Statement of Limited Warranty:
Northwest Life Science Specialties, LLC (NWLSS) makes no guarantee ofany kind, expressed or implied, that extends beyond the description of thematerial in this kit, except that they will meet our specifications at thetime of delivery. Customer's remedy and NWLSS’ sole liability is limited to,at NWLSS’ option, refund of the purchase price, or the replacement ofmaterial not meeting our specification. By acceptance of our product,
customer assumes all liability and will indemnify and hold NWLSS
harmless for the consequence of this product’s use or misuse by the customer, its employees, or others. Refund or replacement is conditioned ofcustomer notifying NWLSS within twenty-one (21) days of the receipt of
product. Failure to give notice within 21 days shall constitute a waiver by
the customer of all claims hereunder with respect to said product.

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Performance Details (continued):
Dynamic Range:
The linearity and sensitivity of the calibration curve defines the usefulrange of the assay. As shown in Figure 5, an unknown sample with a GSH
concentration, following dilution in Assay Buffer, greater than the 20 µM
will not be accurate and should be further diluted and re-assayed.
Dynamic Range

Rate

0.4

0.2

0.0
10

20

Page 5Warnings, Limitations, Precautions:
NADPH
β-Nicotinamide adenine dinucleotide phosphate, reduced form (CAS2646-71-1) is irritating to the eyes, respiratory system and skin. Target
organs: nerves, liver. In case of contact with eyes, rinse immediately withcopious amounts of water and seek medical advice. Wear suitable
protective clothing.
MPA
Metaphosphoric acid (CAS 37267-86-0) is corrosive and may causeburns. In case of contact with eyes, rinse immediately with copious
amounts of water and seek medical advice. Wear protective clothing,
gloves and eye protection.

0.6

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3040

uM GSH

Figure 5.
The calibration curve (─) is compared to the plot of a seriesof GSH concentrations that exceed the range of the assay (■).
Stability
All unopened reagents are stable until the expiration date stated on the
package label when stored at 2-8°C.
Reconstituted NADPH is usable for up to 1 week if stored at 2-8°C and up
to 1 month at -20°C if stored sealed and uncontaminated in the original
bottle.
Deproteinated samples in 5% MPA are stable up to 6 months at -20°C or
indefinitely at -80°C.
Things to Note:
1. Endpoint assay is not recommended; kinetic is more precise andfaster.

DTNB
5-5′-Dithiobis(2-nitrobenzoic acid) (CAS 69-78-3) is irritating to theeyes, respiratory system and skin. In case of contact with eyes, rinse
immediately with copious amounts of water and seek medical advice.
Wear suitable protective clothing.
Interference
Thiol containing compounds, such as cysteine, β-mercaptoethanol or dithiothreitol may compete with GSH for DTNB. N-ethylmaleimide or otherthiol alkylating agents are known to interfere with GR and also will reactwith GSH and should be avoided.
Storage Instructions:
Upon receipt, store the reagents at 2-8ºC. Do not use components beyond
the expiration date printed on the label.
All reagents should be brought to room temperature (18-25ºC) prior touse.
Assay Preparation
Plate Reader Setup Recommendations
Wavelength: 405 nm
Duration: 3 minutes

2. The reagents are provided with a reasonable overfill but use caution if
using multi-channel pipetting troughs to minimize reagent volume loss.
3. Do not pre-combine the NADPH, DTNB and GR. GR will catalyze thereduction of DTNB by NADPH resulting in higher background and/or exhaustion of reagents.

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Interval: 15-20 seconds
Data Reduction: Linear regression

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Reagent Preparation:
5% Metaphosphoric acid
1. Weigh 1 gram of MPA into a beaker or flask
2. Add 20 mL deionized water to the beaker or flask
3. Stir until dissolved
4. Store at 2-8°C until use

Data Analysis: (continued)
Continuing with the example above…
• The unknown rate = 0.197 A405/min• The unknown df (1/3 deproteination and 1/21 buffer dilution) = 63x

Note: 5% MPA solution should be prepared and used the same day.

Substituting the values into Equation 2, the GSH concentration for the
unknown was found to be…

NADPH
1. Add the entire contents of one NADPH Diluent bottle to a
NADPH bottle.
2. Secure cap on vial containing the reconstituted NADPH andmix briefly by inverting the bottle.
Note: Once reconstituted, the NADPH is usable for up to 1 week if
stored at 2-8ºC in the original container. The NADPH reagent
can also be stored at -20°C for up to 6 weeks. However, it isrecommended that the reagent be tested before committing
samples.
Calibrators
Add 50 µL GSH Calibrator to 950 µL Assay Buffer = 20 µM GSH Equiv.
Add 25 µL GSH Calibrator to 975 µL Assay Buffer = 10 µM GSH Equiv.
Assay Buffer Only = 0 µM GSH equivalents.
Other ReagentsThe DTNB, GR and Assay Buffer are supplied ready-to-use.
Sample Handling/Preparation:
The multi-disciplinary interest in measuring GSH has resulted in a myriadof sample types and experimental conditions and is beyond the scope of
this product insert to describe sample processing in detail. However,general guidelines are provided below for representative sample types. If
additional information is required, please contact NWLSS to discuss theparticular sample under investigation.
Deproteination
Most samples will require removal of interfering proteins and metabolizing enzymes prior to assay. The acidic environment of the deproteinatedsample also improves the stability of GSH.

[GSH] =

0.197-0.0091• 63 = 810.8 µM
0.0146

Performance Details:
Precision
The precision of the assay was estimated by measuring a set of controls
having low and high GSH concentrations, in duplicate, two times each day
for five consecutive days using the standard procedure.

ControlµM GSH

Intra-assay

Inter-assay

Mean

s

s

%CV

%CV

Total
s

%CV

Low

219

6

2.7

9

4.1

7

3.2

High

827

11

1.3

14

1.7

12

1.5

Sensitivity
The method detection limit or MLD is estimated using twice the standard
error of the rate of the calibration curve or 0.5 µM GSH. Alternatively, thelower limit of detection (LLD) is defined as 3.29 standard deviations fromzero or 0.1 µM.
Accuracy
Recovery: A 10 mM solution of GSH was diluted in Assay Buffer to 20 µM
and measured against the GSSG calibrators using the standard method.
The GSH recovery was 102% showing the equivalency of the GSSG
calibrators in the assay.

1. Add 50 µL of sample to microcentrifuge tube.
2. Add 100 µL cold 5% MPA.
3. Vortex for 5 seconds (5 count).
4. Centrifuge at >1,000 xg for 5 minutes or equivalent.
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Data Analysis: (continued)
The calibration curve, shown in Figure 4, is constructed by plotting therate for each calibrator as a function of the GSH concentration.
The general equation for the GSH calibration curve is:Rate = a[GSH] + b

Equation 1

where a and b are the slope and intercept of the linear regression
equation, respectively.
Linear regression analysis of the rates as a function of concentration from
Table 1 yielded the following equation:
Rate = 0.01464[GSH] + 0.0091, r2 = 1.000.

Calibration Curve
Rate = 0.01464[GSH] + 0.0091
r2 = 1.000

Rate

0.3

0.2

0.1

0.0

0

5

10

15

20

µM GSH

Figure 4.
Plot of the increase in the rate (∆A405/min) as a functionof the concentration of GSH.
Rearranging Equation 1, the concentration of GSH in the unknown isdetermined given the rate…

[GSH] =

Rate - b• df
aEquation 2

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Deproteination (continued):
5. Carefully remove supernatant.
6. Place supernatant on ice or store at -20°C if sample is to be
assayed at a later date.
7. Just prior to assay, dilute sample at least 1/20 in Assay Buffer
Note: If GSH is to be normalized to hemoglobin or protein, these tests mustbe performed on non-deproteinated samples.
Whole Blood:
Collect samples using EDTA, heparin, citrate or ACD anticoagulant. Store at
2-8°C. DO NOT FREEZE. Deproteinate prior to assay.
TissueThe GSH concentration in most tissues ranges from 1-10 mM;
therefore, a 10% w/v homogenate is recommended. Contaminating blood
contains significant concentrations of GSH and, if practical, should be removed by perfusion with an appropriate buffer, such as phosphate
Buffered saline containing heparin. Clarify homogenates by centrifugationand store the supernatant on ice. Deproteinate as soon as possible toreduce the loss of GSH to various metabolic enzymes and mixed disulfide
reactions. The GSH concentration can be normalized to the wet weight of thetissue sample or to the protein concentration of the homogenate.
Cultured Cells
Prepare a cell suspension of approximately 106 cells per mL in an
appropriate buffer and homogenize or disrupt the cells by sonication. Clarify
the homogenate by centrifugation and store the supernatant on ice. The GSH
concentration can be normalized to the cell number or to the protein concentration of the homogenate. Deproteinate as soon as possible to reduce theloss of GSH to various metabolic enzymes and mixed disulfide reactions. It is
recommended that a trial assay with a representative sample be tested todetermine if the samples are within the dynamic range of the assay. If thetest sample is below the sensitivity of the standard procedure, please contact
NWLSS for assistance.
PlasmaThe GSH concentration in plasma can be at the limits of sensitivity of theassay following standard deproteination and dilution. Therefore, it may benecessary to use the high sensitivity protocol as posted atwww.nwlifescience.com/products/assaykit/gshhighsens.htm.

where df is the dilution factor for the unknown.

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Sample Handling/Preparation (continued):
Oxidized Glutathione
Oxidized GSH (GSSG) can be estimated using the NWLSS GSH Assay by
first scavenging any free GSH in the sample by incubating the homogenate with of 4-vinylpyridine for 60 minutes at room temperature. The concentration of 4-vinylpyridine (4-VP, Sigma V-3877 or equivalent) in thehomogenate should be approximately 10-fold greater than the expected
GSH concentration. Because of measurable interference by 4-VP, thecalibrators must be treated in the same manner as the samples. The
GSSG in most samples is at or below the detection limit of the standard
assay method however a modified protocol for testing GSSG is posted at
http://www.nwlifescience.com/products/PDF/gsh01gssg.htm
for our customers wishing to measure oxidized glutathione.
Assay Protocol:
Standard Procedure
1. Bring all reagents to room temperature.
2. Remove microplate from plastic bag.
3. Add 50 µL of calibrator, diluted sample, and diluted control to a
designated well.
4. Add 50 µL of DTNB to each well.
5. Add 50 µL of GR to each well
6. Incubate microplate for 3-5 minutes at room temperature.
7. Add 50 µL of reconstituted NADPH to each well.
8. Begin recording the absorbance at 405 nm at 15-20 second
intervals for 3 minutes.
9. Determine the concentration of the controls and samples. If
using data reduction on the plate reader, skip steps a-d.
a. Calculate the rate for each calibrator, control andsample from the slope of the linear regression of A405 as afunction of time.
b. Calculate the linear regression parameters to obtain theequation of the line.
c. Calculate the concentrations of the controls and samples.
d. Correct the control and sample for dilution and report results.
Data Analysis:
The following example shows the expected behavior and results of GSH
determination using the standard method with the NWLSS™ Glutathione
assay.
A 50 µL sample of whole blood sample was added to a microcentrifuge
tube containing 100 µL cold 5% MPA. The microcentrifuge tube was thenvortexed, centrifuged and the supernatant collected and placed on ice.
Following the 25:500 dilution in Assay Buffer, the diluted sample was
assayed using the standard method.
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Data Analysis (continued):
The plot of the absorbance as a function of time for each calibrator andthe unknown sample are shown in Figure 3. On inspection the curves arelinear as expected.

Rate Curves
1.4
1.2
1.0

A405

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0.8
0.6
0.4
0.2
0.0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

MinutesFigure 3.
Plot of the A405 as a function of time for calibrators 0(○),
10 µM (x), 20 µM (+) GSH and unknown (■).
Alternatively, the r2 parameter can be used as a measure of linearity(Table 1). If curves appear to be non-linear or have an r2 value less than
0.995, then that sample should be repeated following further dilution,
see Dynamic Range, page 11.
For each curve in Figure 3, the reaction rate (rate = slope) for each curve
was determined using linear regression analysis. The rate for eachcalibrator and the unknown sample are shown in Table 1.

SampleRate∆A405/minr2

Calibrator

0 µM

0.00880.996

Calibrator

10 µM

0.15601.000

Calibrator

20 µM

0.30161.000

0.1972

1.000

Unknown

Table 1: Reaction Rates

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