Nitric Oxide Quantitation Kit

Version 3 – 3-013002-1623(version A4)

Catalog No. 40020

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sample Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Preparation of Nitrate Standard Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Nitrate Assay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Preparation of Nitrite Standard Curve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Nitrite Assay Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Nitrate/Nitrite Recovery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section B. Related Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

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Overview
The Nitric Oxide Quantitation Kit provides a simple and sensitive assay for monitoring nitricoxide production based on nitrate and nitrite determination. The kit uses a two-step assay
method that eliminates the need to use lactate dehydrogenase (LDH). The sensitivity of theassay is equivalent to that of the three-step LDH method and more sensitive than that of thecommonly used two-step assay method. This new method involves the addition of two cofactors to the nitrate reductase reaction. These cofactors accelerate the conversion of nitrate tonitrite while simultaneously degrading excess NADPH to NADP. Thus, the reductase reaction canbe completed within 30 minutes and colorimetric determination can be directly measured by
the addition of Griess Reagent, without the need for lactate dehydrogenase treatment. This kit
is fast and simple and can be applied to the assay of nitrite and nitrate determination in urine,
plasma, serum, saliva, cell lysate and tissue culture medium.
Nitric Oxide Quantitation Kit

FormatCatalog No.

2 x 96-well plates

40020

See the Active Motif products related to the Nitric Oxide assay in Appendix, Section B.

Introduction
Nitric oxide (NO) is a key molecule that, either directly or through intracellular signaling, stimulates host defenses in the immune system, maintains blood pressure in the cardiovascular system
and modulates neural transmission in the brain1. NO is an activator of soluble guanylyl cyclase,which converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), leading to vasodilatation and inhibition of leukocyte and platelet activation2, 3. As the biologically
active component of endothelium-derived relaxing factor, NO has critical roles in the maintenance of vascular homeostasis3. NO acts as a neurotransmitter in the central and peripheral nervous systems and, therefore, is critical in the pathogenesis of stroke and other neurodegenerative disorders4. As a signal transducer in mammalian systems, NO covalently interacts with target
molecules based on redox potential5. Finally, as a highly reactive chemical, NO directly regulates
the activity of many proteins, such as kinases and proteases6.
Nitric oxide synthase (NOS) catalyses the oxidation of the terminal guanidino nitrogen of theamino acid L-arginine to produce NO and L-citrulline7. Three distinct forms of NOS have been
described: neuronal NOS, endothelial NOS and inducible NOS. Neuronal and endothelial NOS
are regulated by physiological changes in intracellular calcium concentrations, whereas inducible
NOS appears to be regulated in a cell-specific manner. Monitoring cellular NO production is a
useful tool for determining NOS activity. However, the volatile nature of NO makes it unsuitable
for most detection methods.

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1

NO

+

O2-

ONOO-

2 NO

+

O2

N2O4

NO

+ NO2-

N2O3

H+
H2O
H2O

NO3-

+

NO2-

+ NO3-

2 NO2- +H++ 2 H+

2 H+

In the cell, NO undergoes a series of reactions with several molecules present in biological fluids
and is eventually metabolized to nitrite (NO2-) and nitrate (NO3-). Thus, the best index of total
NO production is the sum of both nitrite and nitrate, commonly quantified in a two-step assay.
The first step in the measurement of NO is the conversion of nitrate to nitrite by the use of
NADH or NADPH-dependent nitrate reductase. Subsequently, the converted nitrite can bequantified by the addition of Griess Reagent, which converts nitrite into a purple-colored azo
compound. Accurate concentration of nitrite can be determined by photometric measurementof the colored azo compound. In this two-step assay method, NADPH is commonly used as an
essential cofactor for nitrate reduction. However, excess NADPH interferes with the subsequent
Griess reaction, which limits the sensitivity of the two-step assay. In order to remove this penalizing interference, the two-step assay method has been improved by the addition of an additional step. The three-step assay (LDH assay method) includes a lactate dehydrogenase reaction
after the reductase reaction in order to degrade the excess NADPH. Although the additional step
improves the sensitivity when compared to the two-step assay, the LDH assay is tedious andtime-consuming. By the addition of two cofactors to the two-step assay, Active Motif’s Nitric
Oxide Quantitation Kit combines high sensitivity and fast processing of samples in a two-step
assay method (Figure 1).
NitrateNitrate reductase+ Cofactors

Nitric oxide
NitriteAssay using
Griess Reagent

Figure 1: The principle of the Nitric Oxide Quantitation Kit. Nitric oxide is converted to nitrate and nitrite in the cell.
Nitrate in the sample is converted to nitrite in presence of nitrate reductase and cofactors. Then, nitrite is assayed using
Griess Reagent.

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2

Kit Performance and Benefits
The Nitric Oxide Quantitation Kit is for research use only. Not for use in diagnostic procedures.
Assay Time:

Nitrite assay: 30 minutes
Nitrate assay: 90 minutes

Detection Limit:< 1 µM of nitrite/nitrate

Range of Detection:

This procedure provides quantitative results from 2 to 55 µM ofnitrite/nitrate.

Kit Contents
Kit components can be stored at 4°C for 6 months. Upon reconstitution, the Nitrate Reductase
and Cofactors should be stored at -20°C. Refer to the table below and the section Buffer
Preparation and Recommendations in this manual for further details.
Reagent

Quantity

Storage/Stability

10X Assay Buffer

30 ml

4°C for 6 months

Nitrate Reductase

4 vials

Lyophilized: 4°C for 6 months
Reconstituted: -20°C for 2 months

Cofactors

4 vials

Lyophilized: 4°C for 6 months
Reconstituted: -20°C for 2 months

Nitrate Standard

2 x 1.5 ml

4°C for 6 months

Nitrite Standard

2 x 1.5 ml

4°C for 6 months

Griess Reagent A

12 ml

4°C for 6 months

Griess Reagent B

12 ml

4°C for 6 months

96-well plates

2

Plate sealers4

Additional materials required•

Distilled water (HPLC-grade is recommended)•

Multi-channel pipettor•

Rocking platform (for nitrate assay only)•

10,000 Dalton micropore filter (for samples rich in proteins)•

Microplate spectrophotometer with a 540 nm filter for samples and a 620 nm filter
for reference

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3

Protocol
Buffer Preparation and Recommendations
Preparation of 1X Assay Buffer
Prepare the amount of 1X Assay Buffer required for the assay as follows: For every 10 ml of 1X
Assay Buffer required, dilute 1 ml of 10X Assay Buffer in 9 ml of distilled water. This should be
used for reconstitution of Nitrate Reductase, dilution of samples and preparation of standard
curves for nitrate and nitrite assays. Excess 1X Assay Buffer can be stored at 4°C for one week.
Preparation of Nitrate Reductase
The Nitrate Reductase is provided lyophilized in four vials. Each vial is sufficient for 50 reactions.
Reconstitute the contents of each vial with 0.6 ml ice-cold 1X Assay Buffer. Store at –20°C .
Freezing and thawing of reconstituted Nitrate Reductase should be limited to one time.
Preparation of Cofactors
The Cofactors are provided lyophilized in four vials. Each vial is sufficient for 50 reactions.
Reconstitute the contents of each vial with 1.2 ml ice-cold distilled water. Store at –20°C .
Freezing and thawing of the reconstituted Cofactors should be limited to one time.
Preparation of Nitrate Standard
This vial contains a solution of 200 µM NaNO3. Do not add water or Assay Buffer to this vial. The
nitrate standard should be stored at 4°C.
Preparation of Nitrite Standard
This vial contains a solution of 200 µM NaNO2. Do not add water or Assay Buffer to this vial. The
nitrite standard should be stored at 4°C.
Preparation of Griess Reagent
The Griess Reagent is composed of two solutions: Griess Reagent A and Griess Reagent B. Do
not add water or Assay Buffer to these solutions. Store at 4°C.

Sample Preparation and Recommendations
We recommend measuring the nitrate or nitrite concentration in the samples in duplicate.
Urine
Urine may be used directly after proper dilution with 1X Assay Buffer. In some cases, precipitation will occur after the addition of Griess Reagent. If this happens, the sample should be filtered through a 10,000 Dalton micropore filter (e.g. a Millipore UFC801008) prior to assay.

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4Plasma, Serum and Saliva
Citrate, EDTA or heparin may be used as an anticoagulant for the collection of plasma. All samples require at least a 2-fold dilution with 1X Assay Buffer followed by centrifugation to remove
any particles. After the above treatment, samples must be filtered through a 10,000 Dalton
micropore filter (e.g. a Millipore UFC801008) prior to assay.
Culture MediaThe use of a media containing high levels of intrinsic nitrate/nitrite (e.g. RPMI) must be avoided
for cell culture. We recommend adapting the cells to a media poor in nitrate/nitrite (e.g. MEM),
as well as phenol red. Cellular nitrite/nitrate production can be quantified by subtracting thelevel of nitrite/nitrate present in the media from the nitrite/nitrate level present during cell
growth. The effect of components in the media on color development can be addressed by preparing nitrate or nitrite standards in the presence of a fixed volume of the culture media.
Cell Lysate
Cell lysate contains a high number of large proteins that can interfere with the nitrate reductase
or Griess reaction. The sample should be filtered through a 10,000 Dalton micropore filter prior
to the assay to remove high molecular weight proteins.

Preparation of Nitrate Standard Curve
Prior to starting the nitrate assay, make sure that all of the reagents needed are prepared andcooled on ice. For your samples, we recommend using duplicate wells for measuring the nitrateconcentration, whereas one well can be used for each nitrate standard concentration.
The standard curve for nitrate is prepared by the addition of reagents to the plate wells in thefollowing way:
Well

Nitrate Standard (µl)

1X Assay Buffer (µl)

Final Nitrate
Concentration (µM)*

A1

0

70

0

B1

5

65

5

C1

10

60

10

D1

15

55

15

E1

20

50

20

F1

25

45

25

G1

30

40

30

H1

35

35

35

* Concentrations indicated are for those in the final 200 µl assay volume, after addition of the Griess Reagent.

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5

Nitrate Assay Procedure1.

Add 70 µl 1X Assay Buffer to the blank wells.

2.

Add up to 70 µl sample or diluted sample to the sample wells. If the sample volume is lessthan 70 µl, adjust the final volume to 70 µl using the 1X Assay Buffer.
Note:

The pH of the sample must be approximately neutral as the enzyme is inactivated at high and low pH values. Sample volumes for plasma, serum or tissue
homogenates are recommended to be less than 40 µl.

3.

Add 20 µl reconstituted Cofactors solution to each well (blanks, standards and samples).

4.

Add 10 µl reconstituted Nitrate Reductase solution to each well.

5.

Cover plate and mix on a rocking platform set at 150 rpm for one minute.

6.

Incubate the plate for 30 minutes at room temperature. Avoid light exposure during incubation. It is not necessary to shake the plate during incubation.

7.

Add 50 µl Griess Reagent A to each well.

8.

Immediately add 50 µl Griess Reagent B to each well.

9.

Allow the color to develop for 20 minutes at room temperature.

10. Read absorbance on a spectrophotometer at 540 nm with a reference wavelength of
620 nm.

Preparation of Nitrite Standard Curve
Prior to starting the nitrite determination, make sure that all of the reagents needed are prepared and cooled on ice. For your samples, we recommend using duplicate wells for measuring
the nitrite concentration, whereas one well can be used for each nitrite standard concentration.
Nitrite concentrations can be directly measured without performing the Nitrate Reductase reaction. The standard curve for nitrite is prepared by the addition of reagents to the plate wells in
the following way:
Well

Nitrite Standard (µl)

1X Assay Buffer (µl)

Final Nitrite
Concentration (µM)*

A1

0

100

0

B1

5

95

5

C1

10

90

10

D1

15

85

15

E1

20

80

20

F1

25

75

25

G1

30

70

30

H1

35

65

35

* Concentrations indicated are for those in the final 200 µl assay volume, after addition of the Griess Reagent.
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6

Nitrite Assay Procedure1.

Add 100 µl 1X Assay Buffer to the blank wells.

2.

Add up to 100 µl sample to the sample wells. If the sample volume is less than 100 µl, adjust
the final volume to 100 µl using the 1X Assay Buffer.

3.

Add 50 µl Griess Reagent A to each well (blanks, standards and samples).

4.

Immediately add 50 µl Griess Reagent B to each well.

5.

Allow the color to develop for 10 minutes at room temperature.

6.

Read absorbance on a spectrophotometer at 540 nm with a reference wavelength of
620 nm.

Calculations
Subtract the blanks
Average the absorbance values of the sample wells. Subtract the blank value from the meanvalues of the samples.

Plot the standard curves
Plot absorbance as a function of nitrate or nitrite concentration. The nitrate assay converts
nitrate to nitrite, then measures total nitrite. Therefore, the nitrate standard curve is used for
determination of total nitrate and nitrite concentration in the sample, whereas the nitrite standard curve is used only for the nitrite concentration. The nitrate and nitrite standard curves aresupposed to be identical; in practice, however, a small discrepancy often occurs.

Determine nitrate and nitrite concentrations
The standard curves are used as follows:1.

Measure the endogenous nitrite concentration of a sample using the nitrite assay procedure and the standard curve of nitrite.

2.

Measure the total nitrate/nitrite concentration of a sample using the nitrate assay procedure and the standard curve of nitrate.

3.

Determine the endogenous nitrate concentration of a sample by subtracting the endogenous nitrite concentration obtained in (1) from the total nitrate/nitrite concentration
obtained in (2).

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7

Typical Standard Curves
Nitrite Standard Curve
0. 8
y = 0.022x + 0.015

y = 0.022x + 0.007

r 2 = 0.999

0.6

OD540nm

0. 6

OD540nmNitrate Standard Curve

0.8

r 2 = 0.999

0. 4

0.4

0.2

0. 2

0

0
0

10

20

30

0

40

Nitrite concentration (µM)

10

20

30

40

Nitrate concentration (µM)

These nitrate and nitrite standard curves are provided for demonstration only. Standard curves
should be generated for each set of samples assayed.

Nitrate/Nitrite Recovery
Samples may contain proteins or metal ions that could alter the nitrate reductase and/or Griess
reactions. Redox agents such as ascorbic acid, dithiothreitol and mercaptoethanol will interferewith color development. Phosphate concentrations higher than 50 mM will alter the nitrate
reductase activity. In these cases, nitrate and nitrite concentrations in the samples would be
biased. The percentage of nitrate and nitrite recovery from a sample can be determined by adding a fixed amount of nitrite and nitrate standards to the sample, as shown below. Then, use kit
procedures to determine nitrate and nitrite concentrations in samples.
Typical recovery data
A human urine sample was collected, centrifuged and filtered through a 10,000 Dalton micropore
filter. The amount of nitrate in various volumes of the filtrate (0-5 µl) was measured in the presence or absence of exogenous nitrate (final concentration: 10 µM) by the nitrate assay procedure.
The percentage of recovery is determined according to the following procedure:
1.

Subtract the absorbance of the sample containing exogenous nitrate from the absorbanceof the sample without exogenous nitrate.

2.

Divide this value by the absorbance obtained with the 10 µM nitrate standard and multiply
by 100.

3.

Divide the nitrate/nitrite concentration of the sample determined from the corresponding
standard curve by the percentage of recovery corresponding to the amount of sample used
for the assay.

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8

Recovery of Nitrate in Urine
1.5

OD540 nm1
no exogenous nitrate+ 10 µM nitrate

0.5

0
0

1

2

3

4

5

6

Urine Volume (µl)

Urine (µl)

OD540 without nitrate

OD540 with 10 µM nitrate% Recovery

0

0

0.423

100

1

0.180

0.584

95.5

2

0.342

0.729

91.5

3

0.486

0.860

88.4

4

0.636

0.990

83.7

5

0.775

1.121

81.8

Example for the 1 µl urine sample:
1.

Subtract the OD540 with 10 µM nitrate from the OD540 without nitrate.
0.584 - 0.180 = 0.404

2.

Divide this value by the OD540 with 10 µM nitrate in absence of urine.
0.404/0.423 = 0.955

3.

Multiply this number by 100 to obtain the percentage of recovery.
0.955 x 100 = 95.5%

4.

Divide the [nitrate] determined by the nitrate standard curve for OD540 without nitrate by
the percentage of recovery obtained for 1 µl urine.
7.86 x 100/95.5 = 8.23 µM

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9References
1.
2.
3.
4.
5.
6.
7.

Moncada S., Palmer R.M. and Higgs E.A. (1991) Pharmacol. Rev. 43: 109-42.
Arnold W.P., Mittal C.K., Katsuki S. and Murad F. (1977) Proc. Natl. Acad. Sci. USA 74: 3203-7.
Lloyd-Jones D.M. and Bloch K.D. (1996) Annu. Rev. Med. 47: 365-75.
Jaffrey S.R. and Snyder S.H. (1995) Annu. Rev. Cell. Dev. Biol. 11: 417-40
Nathan C. and Xie Q. (1994) Cell 78: 915-8.
Schreck R. and Bauerle P.A. (1991) Trends Cell Biology 1: 39.
Palmer, R.M.J., Ashton, D.S. and Moncada S. (1988) Nature, 333: 664-666.

Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

Erratic values or dispersionof duplicates

Poor pipetting technique

Use a multi-channel pipettor when possible andmake sure it is calibrated

Bubbles in well

Make sure there are no bubbles in the wells prior
to absorbance reading

Incorrect addition of reagentsMake sure the correct amount of reagents are addedin the right orderNo color development in thenitrate standard curve

Incorrect addition of reagents

Repeat assay making sure the correct volumes ofreagents are added to all wells

Nitrate standard curve is not
linear at high concentrationof nitrate

Concentration of nitrate ismore than 55 µM

Use the curve in the linear portion at lower nitrateconcentrations

Enzyme and Cofactor activity is Avoid freeze and thawing of the enzyme andlost due to repeated freezing
Cofactors, and make sure the kit has not expired
and thawing

Nitrite standard curve is not
linear at high concentrationof nitrite

Plate reader settings not
optimalVerify the wavelength and filter settings in theplate reader

Plate reader settings not
optimalVerify the wavelength and filter settings in theplate reader

Nitrite concentration is
too high

Use the curve in the linear portion at lower nitrite
concentrations

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10Section B. Related products
TransAM™ Kits

FormatCatalog No.

TransAM NFκB p65

1 x 96-well plate5 x 96-well plates

40096
40596

TransAM NFκB p50

1 x 96-well plate5 x 96-well plates

41096
41596

TransAM CREB

1 x 96-well plate5 x 96-well plates

42096
42596

TransAM pCREB

1 x 96-well plate5 x 96-well plates

43096
43596

TransAM AP-1 c-Fos

1 x 96-well plate5 x 96-well plates

44096
44596

TransAM AP-1 FosB

1 x 96-well plate5 x 96-well plates

45096
45596

TransAM AP-1 c-Jun

1 x 96-well plate5 x 96-well plates

46096
46596

TransAM HIF-1

1 x 96-well plate5 x 96-well plates

47096
47596

TransAM PPARγ

1 x 96-well plate5 x 96-well plates

40196
40696

TransAM p53

1 x 96-well plate5 x 96-well plates

41196
41696

Application

FormatCatalog No.

WB, IP
WB, IP
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB
WB, IP
WB
WB
WB
WB

100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg100 µg

40924
40925
40929
40930
40931
40932
40922
40923
40926
40933
40934
40935
40927
40938
40939
40940
40941
40936
40937
40963
40964

Antibodies
Caspase 3 mAb
Pro-Caspase 3 mAb
Caspase 7 mAb
Caspase 8 mAb
Caspase 9 pAb
Caspase 14 mAb
I-FLICE pAb
DRAK1 pAb
DRAK2 pAb
DcR3 mAb
DR4 mAb
DR5 mAbDR6 pAbOPG mAbTRADD mAb
TRANCE/RANKL/OPGL mAb
D4-GDI mAb
TAC1 pAb
TROY pAb
DAP-3 mAb
DAP-5 mAb
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11Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:+32 (0)2 653 0001
Fax:
+32 (0)2 653 0050
E-mail:eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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