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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
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Assay kit useful for the rapid and accurate measurement of nitric oxide synthase (NOS) activity. This colorimetric assay uses lactate dehydrogenase (LDH) to destroy excess NADPH (cofactor for NOS) which interferes with the chemistry of Griess Reagent, commonly used for nitrite/nitrate detection. Do not use with nitrate- or nitrite-containing tissue culture media such as RPMI.
Catalogue Number
482702
Brand Family
Calbiochem®
Synonyms
NOS Assay Kit, Colorimetric
Materials Required but Not Delivered
• A plate reader with 540 nm filter • An adjustable pipettor • Glass distilled water or HPLC-grade water
References
References
Nims, R.W., et al. 1995. Methods Enzymol.7, 48. Moncada, S. 1992. Acta Physiol. Scand.145, 201. Nathan, C. 1992. FASEB J.6, 3051. Green, L.C., et al. 1982. Anal. Biochem.126, 131.
Product Information
Detection method
Colorimetric
Form
96 Tests
Format
96-well plate
Kit contains
Assay Buffer, Nitrate Reductase, Cofactors, Nitrate Standard, Lactate Dehydrogenase, Griess Reagents (R1 and R2), Microtiter Plate, Plate Cover, and a user protocol.
Upon arrival store the Microplate at room temperature and all other components of the kit at -20°C.
Background
Nitric oxide (NO), produced in trace quantities by neurons, endothelial cells, platelets, neutrophils and other cells, acts as a unique second messenger molecule. It readily diffuses through cell membranes to exert a variety of biological actions in mammalian cells. Excess generation of NO leads to the formation of peroxynitrite, destruction of iron-sulfur clusters, thiol nitrosylation, and nitration of protein tyrosine residues. The final products of NO in vivo are nitrite (NO2-) and nitrate (NO3-). The relative proportions of these two products are variable. Hence, the best index of total NO produced is the sum of both [NO2-] and [NO3-].
In biological systems, NO is synthesized from L-arginine by the action of nitric oxide synthase (NOS). NADPH is an essential co-factor for the action of this enzyme. Unfortunately, it interferes with the chemistry of the Griess reagents, commonly used for nitrite detection, and leads to lower color yield. This limitation can be easily overcome by either using very small amounts of NADPH (in conjunction with a catalytic system for recycling of the NADP+ to NADPH), or employing an additional step to remove or destroy the excess NADPH. The Calbiochem® brand kit uses lactate dehydrogenase (LDH) to destroy excess NADPH. This kit is well suited for high throughput screening applications. It can be used for the assay of nitrate and nitrite in urine, plasma, serum, tissue culture medium, and tissue homogenates.
• A plate reader with 540 nm filter • An adjustable pipettor • Glass distilled water or HPLC-grade water
Precautions and recommendations
Pipetting Tips
a) To maintain precise times of incubation and for saving time, use of a repipettor is recommended. b) Always use different tips for pipetting assay buffer, standards, samples and color development reagents. c) Before pipetting, equilibrate the pipette tip in the reagent to be used (i.e., fill the tip and expel the solution, repeat a couple of times). d) Do not touch the pipette tip to the reagents already in the well.
Assay Considerations
The assay conditions for NOS can be set up as required by your experimental design. However, the following parameters should be taken into account.
• Sample volume: The amount of sample utilized in the assay for nitrate and nitrite can vary from <10 to 60 µl, depending upon the activity of the enzyme. A convenient amount of sample for each assay is 60 µl. Based on this amount of sample, a total volume of reaction mixture should be at least 200 µl to allow for duplicate or triplicate assays. • Stopping the NOS reaction: The NOS reaction must be stopped by heat inactivation. The use of any acid to terminate the reaction will lead to erroneous results as NO will be released from nitrite under acidic conditions. Following heat inactivation, centrifuge the samples to pellet the denatured protein. • Controls: A heat-inactivated, zero-time control should be included for each NOS preparation to serve as a control to measure endogenous nitrate + nitrite. It is also useful in determining the degree of interference caused by any other material present in the sample. • Linearity of the assay: The use of a single time point assay of enzyme activity is valid only when the steady-state reaction is in the linear phase. Typically, this is done by stopping the reaction when <20% of the substrate(s) has been utilized. • NOS stability: Another concern is the stability of the enzyme for extended periods of time. NOS is not a stable enzyme and its activity may decrease over time at 37°C. The linearity of the assay can be assessed by measuring activity at several time points under identical reaction conditions (example, every minute for 5 or 10 min) and plotting the absorbance (obtained in the nitrate assay) as a function of time. Once linearity has been established, a single time-point assays can be performed. • Interfering substances: Antioxidants will interfere with the color development reaction. Azide, ascorbic acid, dithiothreitol and mercaptoethanol interfere with color development and cause quenching when present at concentrations as low as 100 µM. Alkyl amines, most sugars, lipids or amino acids (except those containing thiol groups) do not interfere. Phosphate concentrations greater than 50 mM will interfere with the conversion of nitrate to nitrite.
Preparation
Nitrate and nitrite are the stable end products of the reaction of nitric oxide (NO) with molecular oxygen. This kit is designed for measuring total nitrate and nitrite content in biological fluids and that derived from NO produced by nitric oxide synthase under controlled assay conditions.
NOS Assay:1. Urine Samples Urine samples can be used directly after dilution to a proper concentration in assay buffer. Urine contains relatively high levels of nitrate (200-2000 µM), therefore, a dilution of approximately 1:10 to 1:50 may be required.
2. Culture Medium Culture media, such as RPMI 1640, may contain high levels of nitrate. It is best not to use this type of media, particularly when small changes in nitrate levels are measured. If it is absolutely necessary to use this type of medium, then cellular nitrate/nitrite levels can be quantitated by subtracting the level of nitrate/nitrite in the medium (in the absence of cells) from the total levels recorded. Cellular nitrate/nitrite production can be quantitated by subtracting the level of nitrate/nitrite present in the media (in the absence of cells) from the total nitrate/nitrite level present during cell growth. The effect of media components on color development can be assessed by making a nitrate standard curve in the presence of a fixed volume of the culture media (usually 30 ml works well) and comparing it to a nitrate standard curve made in buffer alone.
3. Plasma and Serum SamplesNote: Do not use heparinized plasma. Ultrafilter plasma or serum samples through a 10 to 30 kDa cut-off filter using a commercially available centrifuge or microfuge ultrafiltration device. The filters, supplied through Amicon or Millipore, should be rinsed with HPLC-grade water prior to ultrafiltration of serum or plasma. Ultrafiltration reduces absorbance due to the presence of hemoglobin and improves color formation with Griess reagents. Assay for nitrate and/or nitrite using a maximum of 40 µl of the filtrate. Heparinized plasma may form a precipitate upon addition of Griess Reagent R1, thereby making the sample unusable. Citrate or EDTA are recommended as anticoagulants.
4. Tissue Homogenates Homogenize tissues in phosphate buffered saline (PBS), pH 7.4, and centrifuge at 10,000 x g for 20 min. Ultracentrifuge the supernatant at 100,000 x g for 15 min. Ultrafilter using a 10 or 30 kDa molecular weight cut-off filter by centrifugation. Filtration of the 100,000 x g supernatant through a 0.45 µm filter will increase the ultrafiltration rate. The filters should be rinsed with HPLC-grade water prior to ultrafiltration. Assay the sample for nitrate and/or nitrite using a maximum of 40 µl of the filtrate.
5. Assay for in vitro NOS reactions The conditions for an in vitro NOS assay using purified enzymes or tissue and cell homogenates can be set up as required by your experimental design, but the following parameters should be observed.
a. Sample Volume
The amount of sample utilized in the assay for nitrate + nitrite can vary from ≤10 µl-60 µl, depending on the activity of the enzyme. A convenient amount of sample for each assay will by 60 µl. Based on this amount of sample, a total volume for the NOS reaction should be at least 200 µl to allow for duplicate or triplicate analysis of the products.
b. Stopping the NOS Reaction
Heat inactivation is recommended for stopping the NOS reaction. The use of acid to quench the NOS reaction will lead to erroneous results for two reasons: NO will be released from nitrite under acidic conditions and nitrate reductase is inhibited by a variety of acids even when the pH has been adjusted to neutral. Following heat inactivation, centrifuge the samples to pellet the denatured protein.
Reagent preparation
Reconstitution of the Reagents
Some of the kit components are in lyophilized form and should be reconstituted prior to use. Follow the directions carefully to ensure that proper volumes of water or assay buffer are used to reconstitute vial contents.
Assay Buffer Dilute the contents of the Assay Buffer vial to 100 ml with HPLC-Grade water. This assay buffer should be used for dilution of samples as needed prior to assay.
Nitrate Reductase Reconstitute the contents of the vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store the unused portion at -20°C. Do not freeze/thaw this solution more than once.
Co-factor Preparation Reconstitute the contents of the vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store the unused portion at -20°C. Do not freeze/thaw this solution more than once.
Nitrate Standard Remove the vial stopper slowly to minimize disturbance of the lyophilized powder. Reconstitute the contents of the vial with 1 ml of Assay Buffer. Vortex and mix sufficiently to ensure all powder in the vial, including any on the stopper, is in solution. Store at 4°C; do not freeze reconstituted solutions. The reconstituted standard will be stable for about four months when stored at 4°C.
Lactate Dehydrogenase Reconstitute the contents of the vial with 1.2 ml of assay buffer. Keep on ice during use. Aliquot and store the unused portion at -20°C. Do not freeze/thaw this solution more than once.
Griess Reagent R1 and R2 These are ready-to-use solutions. Do not add water or assay buffer to these reagents.
NADPH Prepare a 1 mM solution of NADPH in assay buffer. At least 1 ml of this solution is required for the nitrate assays and must be made fresh each day.
Detailed protocol
Measurement of Nitrate + Nitrite To quantitate the sample nitrate + nitrite concentrations a nitrate standard curve must be performed.
1. Pipette 0.9 ml of the Assay Buffer into a clean test tube. 2. Add 0.1 ml of the reconstituted Nitrate Standard and vortex. This diluted Nitrate Standard will be used for the preparation of the nitrate standard curve. 3. Pipette the following reagents into designated wells on the plate. 4. Pipette 200 µl of water or assay buffer into wells designated as blank. Do not add any other reagents to these wells. 5. Pipette up to 60 µl of sample into the wells (40 µl for plasma, serum or tissue homogenates). The final volume must be adjusted to 60 µl using the assay buffer solution. 6. Add 10 µl of the freshly prepared NADPH solution (1 mM) to each well (standards and samples). 7. Add 10 µl of the nitrate reductase solution to each well (standards and samples). 8. Incubate at room temperature for 40 min (60 min for plasma, serum, and tissue homogenates). 9. Add 10 µl of the cofactors solution and 10 µl of the LDH solution to each well (standards and samples). 10. Incubate at room temperature for 20 min. 11. After the required incubation time, add 50 µl of Griess reagent R1 to each well (standards and samples). 12. Immediately add 50 µl of Griess reagent R2 to each well (standards and samples). 13. Allow the color to develop for 10 min at room temperature. 14. Read the absorbance at 540 nm using the plate reader. Use the blank wells to zero the instrument.
Standard curve
Plot the absorbance at 540 nm against nitrate concentration and determine the equation of the line. The slope of the line is essentially the extinction coefficient of the product formed from the reaction of nitrite with Griess reagents R1 and R2.
Figure 2: Standard Curve
Determination of sample nitrate or nitrite concentrations
Table 1: Diluted Nitrate Standard
The addition of other reagents to the standard curve are detailed in the next section.
Sensitivity Notes
When using the maximum amount of sample for the nitrate assay (60 µl), the detection limit is 2.5 µM. The detection limit for plasma is higher and only 40 µl of sample is sufficient.
Plate configuration
There is no specific pattern for using the wells on the plate. However, it is necessary to have some wells (at least 2) designated as absorbance blanks (containing 200 µl of assay buffer or water). Their absorbance value must be subtracted from the absorbance measured in all of the other wells. A standard curve for nitrate should also be included in the experimental design and sufficient number of wells should be assigned for this.
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Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
