OXFORD BIOMEDICAL RESEARCH

P.O. Box 522, Oxford MI 48371 USA

USA: 800-692-4633 Fax: 248-852-4466
www.oxfordbiomed.com
NB88.050114 Page 1 of 6
_____________________________________________________________________________________________________________________
Colorimetric Non-enzymatic Assay for
Nitric Oxide
Product No. NB 88
For Research Use Only
_____________________________________________________________________________________________________________________
INTRODUCTION
_____________________________________________________________________________________________________________________
This kit allows you to measure total nitric oxide (NO) produced for in vitro experimental systems
following conversion of nitrate to nitrite by metallic cadmium.
Nitric oxide can be spectrophotometrically assayed by measuring the accumulation of its stable
degradation products, nitrate and nitrite. The ratio of these two products in biological fluids, tissue
culture media, etc. may vary substantially. Hence, for accurate assessment of the total nitric oxide
generated, one must monitor both nitrate and nitrite. An excellent solution to this problem involves
the conversion of nitrate to nitrite catalyzed by cadmium. This is then followed by the quantitation of
nitrite using the griess reaction.
This kit is suitable for the quantitative determination of total nitric oxide in samples that have high
protein concentrations. The cadmium catalyst proves far more robust when in the presence of harsh
deproteinating reagents than the alternative, nitrate reductase.
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PRINCIPLE OF ASSAY
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In aqueous solution, nitric oxide rapidly degrades to nitrate and nitrite. Spectrophotometric
quantitation of nitrite using Griess Reagent is straightforward, but does not measure nitrate. This kit
employs metallic cadmium for quantitative conversion of nitrate to nitrite (figure 1) prior to
quantitation of nitrate using Griess reagent thus providing for accurate determination of total NO
production.
Nitric Oxide
Nitrate
Nitrite
Cd
This kit can be used to accurately measure as little as 1 pmol/mL (~1mM) NO produced in aqueous
solutions. Very little sample is required (1 to 100mL depending on the [NO] in the sample). The
completed reaction is read at 540 nm.
_____________________________________________________________________________________________________________________
MATERIALS PROVIDED
_____________________________________________________________________________________________________________________
1. GRANULATED CADMIUM (NB88a): 25g of granulated cadmium (Cd
++
) beads for nitrate
reduction.
2. ZnSO
4
SOLUTION (NB88b): 2mL of 30% (wt/vol) ZnSO
4
for sample deproteination.
NB88.050114 Page 2 of 6
3. MICROCENTRIFUGE TUBES

(NB88c): 50 x 1.5-mL microcentrifuge tubes for overnight
incubation of the samples.
4. COLOR REAGENT #1 (NB88d): 7 mL of Sulfanilamide (p-Aminobenzenesulfonamide)
dissolved in 3N HCl.
5. COLOR REAGENT #2 (NB88e): 7 mL of N-(1-Naphthyl) ethylenediamine dihydrochloride
dissolved in deionized H
2
O.
6. NITRITE STANDARD (NB88f): 1.5 mL 500pmol/mL NaNO
2
(equivalent to 500 mM NO).
7. MICROTITER PLATE (NB88g): One optically clear 96 well microplate.
8. MICROTITER PLATE TEMPLATE (NB88h): One 96 well microplate template.
9. REAGENT RESERVOIRS (NB88i): Three plastic troughs for dispensing and pipetting
reagents.
10. CADMIUM BEAD HCl-WASH SOLUTION (NB88j): 125 mL of 0.1 M HCl.
11. CADMIUM BEAD NH
4
OH-WASH SOLUTION (NB88k): 125 mL of 0.1 M NH
4
OH.
_____________________________________________________________________________________________________________________
MATERIALS NEEDED BUT NOT PROVIDED
_____________________________________________________________________________________________________________________
1. Microplate reader with 540nm filter.
2. Precision pipettes (10 mL 1,000 mL) and disposable tips.
3. Microcentrifuge tubes or equivalent for dilution, transferring and centrifugation of samples.
4. Vortex mixer.
5. Double Deionized Water (DD water).
_____________________________________________________________________________________________________________________
STORAGE CONDITIONS
_____________________________________________________________________________________________________________________
Cadmium should be stored in a dedicated dry environment.
All other kit components should be stored at 4 C. Alternatively, this kit exhibits good stability at RT
for extended periods of time during shipping and preparations.
_____________________________________________________________________________________________________________________
WARNINGS AND PRECAUTIONS
_____________________________________________________________________________________________________________________
1. Use aseptic technique when opening and dispensing reagents.
2. This kit is designed to work properly as provided and instructed. Additions, deletions or
substitutions to the procedure or reagents is not recommended as it may be detrimental to the
assay.
3. Exercise universal precautions during the performance or handling of this kit or any component
contained therein.
_____________________________________________________________________________________________________________________
PROCEDURAL NOTES
_____________________________________________________________________________________________________________________
1. Reagents can be used immediately upon removal from refrigeration.
2. Performance of the entire kit at once is not required. When performing this kit in part, please
adhere to the following:
-All unused components should be returned to their respective storage areas.
-Create a standard curve for each performance of the assay. Sufficient stock solution of
standard is provided for two standard curves.
3. Use fresh pipette tips when transferring or pipetting reagents from stock reagents.
4. Pipette tips should be pre-wetted or rinsed prior to dispensing reagent. To do this, adjust pipette
to the volume desired and draw up reagent of choice into the tip 2-3 times before uptake for
dispensing.
NB88.050114 Page 3 of 6
5. To minimize error due to handling, wipe the exterior bottom of the microplate wells with a lint-
free paper towel.
6. Take note of the volume of each sample at the various stages of preparation and assay procedure
to ensure proper dilution factor and NO concentration calculation.
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REAGENT PREPARATION
_____________________________________________________________________________________________________________________
Cadmium beads should be washed just prior to use. Cadmium beads prepared days prior to its
intended use should be stored in a sealed container under inert gas such as nitrogen or argon.
All other kit components are supplied ready to use.
Cadmium Wash Procedure:
1. Place the desired amount of Cd
++
in an appropriately sized, sealable container or containers.
Please consider that for every 0.5 g of Cd
++
there will be 1.0 mL of wash solution applied.
2. Apply 1.0 mL of DD H
2
O for every 0.5 g of Cd to be used. Seal container and mix by
inversion five times.
3. Decant the remaining solution and repeat Step 2 one time prior to proceeding to Step 4.
4. Remove residual amounts of solution with a probing pipette or by dabbing with a lint-free paper
towel.
5. Apply 1.0 mL of HCl-Wash solution for every 0.5 g of Cd
++
to be used. Seal container and mix
by inversion five times.
6. Decant the remaining solution and repeat Step 5 one time prior to proceeding to Step 7.
7. Remove residual amounts of solution with a probing pipette or by dabbing with a lint-free paper
towel.
8. Apply 1.0 mL of NH
4
OH-Wash solution for every 0.5 g of Cd
++
to be used. Seal container and
mix by inversion five times.
9. Decant the remaining solution and repeat Step 8 one time prior to proceeding to Step 10.
10. Remove residual amounts of solution with a probing pipette or by dabbing with a lint-free paper
towel.
11. The washed and dried Cd
++
is now ready for use. If you are not using the washed Cd
++
immediately then store the Cd
++
under inert gas such as nitrogen or argon in a sealed container.
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SAMPLE PREPARATION
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This kit is designed for use with samples possessing high concentrations of protein such as serum,
culture medium, or tissue homogenates. Each sample should be diluted according to its presence of
NO metabolites (nitrate and nitrite). Pilot studies or other research should serve as a precedent for the
optimal dilution.
1. Adjust 10-50 mL of sample volume to 190 mL with DD water.
2. Add 10 mL of 30% (wt/vol) ZnSO
4
to the 190 mL of diluted sample and vigorously mix and
incubate at RT for 15 minutes.
3. Centrifuge sample at 3,000 x g for 5 minutes.
4. Transfer the resulting supernatant to a collection tube for sample storage at 20 C or proceed to
the Sample Incubation Procedure located below.
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SAMPLE INCUBATION PROCEDURE
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1. Place approximately 0.5 g of washed and dried Cd
++
beads in a dedicated centrifuge tube for
each representative sample.
NB88.050114 Page 4 of 6
2. Add the deproteinated and clarified sample directly to dedicated centrifuge tube with the Cd
++
present. The volume of the sample will be approximately 200 mL.
3. Incubate the sample and Cd
++
together for 24 hours.
4. After incubation, transfer the sample to a clean microcentrifuge tube for further clarification by
centrifugation. Centrifuge for 5 minutes at 3,000 x g. Begin your assay within 1 hour for best
results. The used Cd
++
beads should be collected and washed as indicated in the reagent
preparation section above.
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STANDARD PREPARATION
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The stock nitrite standard is provided as a 500 mM NO equivalents (500 pmol/mL of NH
2
). Prepare the
standards according to the following Table 1.
Table 1.
Standard NO
Equivalents
(mM)
DD H
2
O
(mL)
Transfer
Volume
(mL)
Transfer
Source
Final
Volume
(mL)
S
7
100 2.0 0.5 Stock 1.5
S
6
50 0.5 0.5 S
7
0.5
S
5
25 0.5 0.5 S
6
0.5
S
4
10 0.5 0.5 S
5
0.5
S
3
5 0.75 0.5 S
4
0.75
S
2
1 0.5 0.5 S
3
0.5
S
1
0.5 2.0 0.5 S
2
2.5
S
0
0 0.5 --- --- 0.5
NOTE: The prepared standards do not require incubation with cadmium.
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ASSAY PROCEDURE
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1. Establish a plate layout accounting for each sample and standard. See Scheme I for a suggested
plate layout.
2. Add 100 mL of sample and standard to their respective wells on the plate. Note: samples may
require further dilution with DD water if the concentration exceeds the standard curve
parameters.
3. Add 50 mL Color Reagent #1 and shake briefly.
4. Add 50 mL Color Reagent #2. Shake for 5 minutes at room temperature.
5. Read plate at 540 nm and proceed to the calculations section.
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INERPRETATION OF RESULTS
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Sample concentrations can be determined using Y-intercept equation as demonstrated below.
Note: The standard curve is demonstrated in Nitric Oxide mM equivalents but can alternatively be
demonstrated in pmol/mL of NH
2
.
1 mM NO = pmol/mL NH
2
1. Average the O.D. values for each replicate of sample and standard.
2. Plot the standard curve using the standard concentration (X-axis) vs. the corresponding O.D. (Y-
axis).
NB88.050114 Page 5 of 6
3. Determine the concentration of each sample by interpolation from the standard curve using the
Y-intercept equation. Enter a sample O.D. value in place of Y and solve for X to
determine the concentration.
Y=mX+b where:
Y= The ordinate or y-axis value (corresponds to the OD reading).
X= The abscissa or x-axis value (corresponds to the NO concentration).
m= Slope.
b= Y axis intercept.
Typical Standard Curve
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 20 40 60 80 100
Nitric Oxide M
O
D
Scheme I. Suggested Plate Layout
1 2 3 4 5 6 7 8 9 10 11 12
A
S
0
S
0
U
1
U
1
U
9
U
9
U
17
U
17
U
25
U
25
U
33
U
33
B
S
1
S
1
U
2
U
2
U
10
U
10
U
18
U
18
U
26
U
26
U
34
U
34
C
S
2
S
2
U
3
U
3
U
11
U
11
U
19
U
19
U
27
U
27
U
35
U
35
D
S
3
S
3
U
4
U
4
U
12
U
12
U
20
U
20
U
28
U
28
U
36
U
36
E
S
4
S
4
U
5
U
5
U
13
U
13
U
21
U
21
U
29
U
29
U
37
U
37
F
S
5
S
5
U
6
U
6
U
14
U
14
U
22
U
22
U
30
U
30
U
38
U
38
G
S
6
S
6
U
7
U
7
U
15
U
15
U
23
U
23
U
31
U
31
U
39
U
39
H
S
7
S
7
U
8
U
8
U
16
U
16
U
24
U
24
U
32
U
32
U
40
U
40
NB88.050114 Page 6 of 6
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REFERENCE
_____________________________________________________________________________________________________________________
Schmidt, H.H., et. al. Biochemica 2:22-23 (1995).
_____________________________________________________________________________________________________________________
DISCLAIMER
_____________________________________________________________________________________________________________________
This information is believed to be correct but does not purport to be all inclusive and shall be used
only as a guide. Oxford Biomedical Research, Inc. shall not be held liable for any damage resulting
from handling or from contact with the above product. See catalog for additional terms and
conditions of sale.
_____________________________________________________________________________________________________________________
ORDERING INFORMATION
_____________________________________________________________________________________________________________________
For additional kits or a complete catalog please call 1-800-692-4633.
_____________________________________________________________________________________________________________________
TECHNICAL SUPPORT
_____________________________________________________________________________________________________________________
If you need technical information or assistance with assay procedures, please call our Technical
Support Department at 1-800-692-4633 or 1-248-852-8815. Our staff will be happy to answer your
questions about this or any other product in the Oxford Biomedical line.
_____________________________________________________________________________________________________________________
LIMITED WARRANTY
_____________________________________________________________________________________________________________________
Oxford Biomedical Research, Inc. makes no warranty of any kind, expressed or implied, except that
this product will meet our specifications at the time of delivery. Buyers exclusive remedy and
Oxford Biomedical Research Inc.s sole liability shall be limited to the refund of the purchase price
or, at the discretion of Oxford Biomedical Research, Inc., the replacement of all materials that do not
meet our specifications. Oxford Biomedical Research, Inc. shall not be otherwise liable for incidental
or consequential damages including, but not limited to, the costs of handling and shipping.
Oxford Biomedical Research, Inc.
P.O. Box 522
Oxford, MI 48371 U.S.A.
Orders: 800-692-4633
Technical Service: 248-852-8815
Fax: 248-852-4466
E-mail: [email protected]
Made in the U.S.A.