482700 Nitric Oxide Synthase Assay Kit

482700
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Radiometric

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      482700-1KTT
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          Glass bottle 1 ktt
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          This product has been discontinued.



          Assay kit useful for the rapid quantitative measurement of nitric oxide synthase (NOS) activity. It is based on the conversion of radioactive L-arginine to L-citrulline by NOS. Radioactive citrulline is separated from unreacted arginine on a cation-exchange resin and quantitated. Note: It is necessary to check the radiopurity of 3H-arginine before proceeding to use the kit.

          Catalogue Number482700
          Brand Family Calbiochem®
          SynonymsNOS Assay Kit
          Materials Required but Not Delivered 3H-Arginine monohydrochloride (40-70 Ci/mmol, 1 µCi/µl, Amersham, Arlington Heights, Illinois, Cat. No. TRK698) or 14C-Arginine monohydrochloride (~300 mCi/mmol, 50 µCi/ml (Amersham, Arlington Heights, Illinois, Cat. No. CFB63)
          10 mM Tris-HCl, pH 7.4
          Scintillation fluid and vials
          References
          ReferencesBredt, D.S., and Snyder, S.H. 1994. Annu. Rev. Biochem. 63, 175.
          Kobzik, L., et al. 1994. Nature 372, 546.
          Moncada, S., and Higgs, A. 1994. N. Engl. J. Med. 329, 2002.
          Nathan, C., and Xie, Q.W. 1994. J. Biol. Chem. 269, 13725.
          Busconi, L., and Michel, T. 1993. J. Biol. Chem. 268, 8410.
          Marletta, M.A. 1993. J. Biol. Chem. 268, 12231.
          Schmidt, H.H., et al. 1993. Biochim. Biophys. Acta 1178, 153.
          Ignarro, L.J. 1991. Biochem. Pharmacol. 41, 485.
          Pollock, J.S., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 10480.
          Schmidt, H.H., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 365.
          Slater, M., et al. 1991. FEBS Lett. 291, 145.
          Stuehr, D.J., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 7773.
          Bredt, D.S., and Snyder, S.H. 1990. Proc. Natl. Acad. Sci. USA 87, 689.
          Bredt, D.S., and Snyder, S.H. 1989. Proc. Natl. Acad. Sci. USA 86, 9030.
          Knowles, R.G., et al. 1989. Proc. Natl. Acad. Sci. U.S.A 86, 5159.
          Product Information
          Detection methodRadiometric
          Form50 Tests
          FormatScintillation vial
          Kit containsHomogenization Buffer, Reaction Buffer, NADPH, CaCl₂, Calmodulin, Stop Solution, Rat Brain Cerebellum Extract, NG-Nitro-L-Arginine Methyl Ester (L-NAME), Equilibrated Resin, Spin Cups, Cup Holders, and a user protocol. Radioactive arginine is not provided with this kit.
          Applications
          Biological Information
          Assay range2 h
          Sample TypeTissue or cell extracts
          Physicochemical Information
          Sensitivitypicomolar range
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 22-36/37/38

          Harmful if swallowed.
          Irritating to eyes, respiratory system and skin.
          S PhraseS: 26-36

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing.
          Product Usage Statements
          Intended useThe Calbiochem® Nitric Oxide Synthase (NOS) Assay Kit is a simple, sensitive and specific assay for nitric oxide synthase (NOS) activity. The NOS assay kit is based on the biochemical conversion of L-arginine to L-citrulline by NOS. Advantages of the NOS assay kit include the use of radioactive substrates (³H-arginine or ¹⁴C-arginine), and an easy method for separating neutrally charged citrulline from positively charged arginine. This kit has sensitivity to the picomole level and allows the assay of up to fifty samples in less than 2 h.
          Storage and Shipping Information
          Ship Code Multiple Storage Conditions
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage Multiple storage requirements
          Storage ConditionsUpon arrival store the Homogenization Buffer, Stop Buffer, Equilibrated Resin, CaCl₂, Elution Buffer, and Spin Cups and Cup Holders at room temperature; store the Rat Brain Cerebellum Extract, Calmodulin, Reaction Buffer, NADPH, and L-NAME, HCl at -70°C.
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsHomogenization Buffer, Reaction Buffer, NADPH, CaCl₂, Calmodulin, Stop Solution, Rat Brain Cerebellum Extract, NG-Nitro-L-Arginine Methyl Ester (L-NAME), Equilibrated Resin, Spin Cups, Cup Holders, and a user protocol. Radioactive arginine is not provided with this kit.
          Specifications

          Documentation

          SDS

          Title

          Safety Data Sheet (SDS) 

          Certificates of Analysis

          TitleLot Number
          482700

          References

          Reference overview
          Bredt, D.S., and Snyder, S.H. 1994. Annu. Rev. Biochem. 63, 175.
          Kobzik, L., et al. 1994. Nature 372, 546.
          Moncada, S., and Higgs, A. 1994. N. Engl. J. Med. 329, 2002.
          Nathan, C., and Xie, Q.W. 1994. J. Biol. Chem. 269, 13725.
          Busconi, L., and Michel, T. 1993. J. Biol. Chem. 268, 8410.
          Marletta, M.A. 1993. J. Biol. Chem. 268, 12231.
          Schmidt, H.H., et al. 1993. Biochim. Biophys. Acta 1178, 153.
          Ignarro, L.J. 1991. Biochem. Pharmacol. 41, 485.
          Pollock, J.S., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 10480.
          Schmidt, H.H., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 365.
          Slater, M., et al. 1991. FEBS Lett. 291, 145.
          Stuehr, D.J., et al. 1991. Proc. Natl. Acad. Sci. U.S.A 88, 7773.
          Bredt, D.S., and Snyder, S.H. 1990. Proc. Natl. Acad. Sci. USA 87, 689.
          Bredt, D.S., and Snyder, S.H. 1989. Proc. Natl. Acad. Sci. USA 86, 9030.
          Knowles, R.G., et al. 1989. Proc. Natl. Acad. Sci. U.S.A 86, 5159.

          Brochure

          Title
          Nitric Oxide and Oxidative Stress Brochure
          User Protocol

          Revision01-April-2011 RFH
          SynonymsNOS Assay Kit
          Form50 Tests
          FormatScintillation vial
          Detection methodRadiometric
          StorageUpon arrival store the Homogenization Buffer, Stop Buffer, Equilibrated Resin, CaCl₂, Elution Buffer, and Spin Cups and Cup Holders at room temperature; store the Rat Brain Cerebellum Extract, Calmodulin, Reaction Buffer, NADPH, and L-NAME, HCl at -70°C.
          Intended useThe Calbiochem® Nitric Oxide Synthase (NOS) Assay Kit is a simple, sensitive and specific assay for nitric oxide synthase (NOS) activity. The NOS assay kit is based on the biochemical conversion of L-arginine to L-citrulline by NOS. Advantages of the NOS assay kit include the use of radioactive substrates (³H-arginine or ¹⁴C-arginine), and an easy method for separating neutrally charged citrulline from positively charged arginine. This kit has sensitivity to the picomole level and allows the assay of up to fifty samples in less than 2 h.
          BackgroundMeasuring NOS activity by monitoring the conversion of arginine to citrulline is currently the standard assay for NOS activity in both crude and purified enzyme preparations. This reaction involves a five-electron oxidation of a guanidinonitrogen of L-arginine to nitric oxide (NO), together with the stoichiometric production of L-citrulline (see Figure 1). The reaction consumes 1.5 equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and also requires molecular oxygen, calcium, calmodulin and tetrahydrobiopterin.



          Figure 1: Principle of the Assay

          NOS catalyzes a 5-electron oxidation of an amidine nitrogen of L-arginine to generate NO and L-citrulline. L-Hydroxy-arginine is formed as an intermediate that is tightly bound to the enzyme. Both steps in the reaction are calcium and calmodulin dependent.

          Principles of the assayThe Calbiochem® Nitric Oxide Assay Kit allows for the routine assay of NOS using radioactive arginine, which is added to tissue or cell extracts. After incubation, the reactions are stopped with a buffer containing EDTA, which chelates the calcium required by NOS and, consequently, inactivates the NOS. Equilibrated resin, which binds to arginine, is added to the samples and they are then applied to spin cups. The citrulline, being ionically neutral at pH 5.5, passes through the column completely. The NOS activity is then determined by quantitating the radioactivity in the eluate.
          Materials provided• Homogenization Buffer (Kit Component No. KP1101-50ML: 1 bottle, 50 ml, 250 mM Tris-HCl, 10 mM EDTA, 10 mM EGTA, pH 7.4; supplied as 10X
          • Stop Buffer (Kit Component No. KP1102-25ML): 1 bottle, 25 ml, 50 mM HEPES, 5 mM EDTA, pH 5.5
          • Equilibrated Resin (Kit Component No. KP1103-5ML): 1 vial, 5 ml
          • Calcium Chloride (CaCl₂) (Kit Component No. KP1104-500UL): 1 vial, 500 µl, supplied as 6 mM
          • Elution Buffer (Kit Component No. KP1105-20ML): 1 bottle, 20 ml, 0.5 M NH₄Cl
          • Spin Cups and Cup Holdersb (Kit Component No. KP1106-1EA):
          • Cerebellum Extract, Rat Braina (Kit Component No. KP1107-20UL): 5 vials, 20 µl each
          • Calmodulin (Kit Component No. KP1108-250UL): 1 vial, 250 µl, supplied as 1 µM
          • Reaction Buffer (Kit Component No. KP1109-1.25ML: 1 vial, 1.25 ml, 50 mM Tris-HCl, 6 µM Tetrahydrobiopterin, 2 µM FAD, 2 µM FMN, pH 7.4; supplied as 2X
          • NADPH, Tetrasodium Salt (Kit Component No. KS0108-25MG): 1 vial, 25 mg
          • NG-Nitro-L-arginine Methyl Ester, HCl, (L-NAME, HCl) (Kit Component No. KP1111-40UL): 1 vial, 40 µl

          a This positive control is a homogenate of rat brain tissue resuspended in Homogenization Buffer.
          b Sufficient spin cups and cup holders are provided for 50 reactions.
          Materials Required but not provided 3H-Arginine monohydrochloride (40-70 Ci/mmol, 1 µCi/µl, Amersham, Arlington Heights, Illinois, Cat. No. TRK698) or 14C-Arginine monohydrochloride (~300 mCi/mmol, 50 µCi/ml (Amersham, Arlington Heights, Illinois, Cat. No. CFB63)
          10 mM Tris-HCl, pH 7.4
          Scintillation fluid and vials
          PreparationPreparation of Extracts from Tissues and Cultured Cells Note: The citrulline assay has been used to quantify levels of NOS activity in tissue homogenates from numerous sources including blood vessels, immune cells, visceral organs, nervous tissue and cultured cells. Nitric oxide synthase is relatively unstable; therefore, tissues should be harvested quickly after animal euthanasia. If enzyme assays are to be performed at a later time, it is best to freeze intact tissues or harvested cultured cells prior to homogenization. Wrap the tissues in aluminum foil, flash freeze in liquid nitrogen and then store at -80°C. Extraction of Proteins from Tissues 1. Add 20 ml ice-cold 1X Homogenization Buffer per gram of tissue. 2. Homogenize the tissue using a tissue grinder or an equivalent tissue homogenizer. Keep tissue homogenate on ice. 3. Distribute the tissue homogenate into 1 ml aliquots in microcentrifuge tubes and spin the tubes in a microcentrifuge at full speed for 5 min at 4°C. 4. Transfer the supernatants to fresh microcentrifuge tubes and keep the tubes on ice until use. Subcellular Tissue Distribution The subcellular distribution of NOS is tightly regulated in tissues. Endothelial NOS (eNOS or NOS-III) is largely membrane associated as a result of N-terminal myristoylation. nNos is found primarily in the cytoplasmic fractions in adult rat brain, yet in skeletal muscle, it is predominately associated with membrane fraction. Nitric oxide synthase in soluble and membrane-associated fractions can be separated by centrifuging the homogenized tissues at 100,000 x g for 60 min. The supernatant contains soluble NOS, while the pellet, which is resuspended in homogenization buffer, contains membrane-associated NOS. Extraction of Proteins from Tissue Culture Cells Certain cultured cells, such as endothelial cells and activated macrophages, contain NOS that can be measured using the citrulline assay. The proteins must first be extracted from the cells as follows: 1. Wash the tissue culture cells once with phosphate-buffered saline (PBS). Harvest the cells in PBS containing 1 mM EDTA, and transfer them to microcentrifuge tubes. 2. Pellet the cells by spinning in a microcentrifuge at full speed for 2 min at 4°C. 3. Remove the supernatant from the pelleted cells by vacuum aspiration. Resuspend the pellet in 100-500 µl of 1X Homogenization Buffer and disrupt the cells by repeated pipetting. 4. Spin the homogenates in a microcentrifuge at full speed for 5 min. 5. Separate the supernatant from the pellet and use supernatant and/or pellet for NOS assay (depending upon the known distribution of NOS). Protein concentration in the range of 5-10 mg/ml in recommended for this assay.
          Reagent preparation• 1X Homogenization Buffer: Prepare 1X Homogenization Buffer by making a 1:10 dilution of Homogenization Buffer in dH2O • Stability and Purification of Radiolabeled Arginine: Prior to initiating the enzyme assays, it is essential to verify the purity of the radiolabeled arginine; otherwise, the resistant high blank value will greatly reduce the sensitivity of the assay. To assess the blank value, a reaction mixture is applied to the equilibrated resin. Non-adherent radioactivity is eluted with stop buffer, the eluate is collected and the radioactivity is quantified in a scintillation counter. Stability of Radiolabeled Arginine 1. Prepare the reaction mixture by combining the following components and place on ice.

          Table 1: Reaction mix

          2. Prepare a reaction sample by combining 100 µl of well-resuspended equilibrated resin and 10 µl of the above reaction mixture in a microcentrifuge tube. 3. Add 400 µl of stop buffer to the reaction sample, mix, and transfer the reaction sample to a spin cup. Place the spin cup into a spin cup holder. 4. Centrifuge the spin cup and holder in a microcentrifuge at full speed for 30 s. 5. Collect 100 µl of the eluate from the spin cup, add scintillation fluid, and quantify the radioactivity in a liquid scintillation counter. Greater than 95% of the applied radioactivity should be retained by the spin cup. This represents a relatively low blank value. If more than 5% of the radioactivity flows through the spin cup, it is important to purify the arginine prior to conducting the assay. 3H-Arginine is prone to radiolytic decay and must be purified every 2 months, while 14C-Arginine is more stable but much more expensive. Purification of Radiolabeled Arginine Radioactive arginine can be purified with the equilibrated resin included in the NOS assay kit as follows: 1. Apply the radioactive arginine to 0.5 ml of equilibrated resin in a disposable spin column [e.g., a Poly-Prep® chromatography column (Bio-Rad, Richmond, California, Catalog #731-1550)]. 2. Wash the column with 5 ml of distilled water. 3. Elute the arginine with 2 ml washes of elution buffer. 4. Lyophilize the arginine and resuspended the arginine in 2% (v/v) ethanol.
          Detailed protocolMeasurement of Nitric Oxide Synthase Activity

          Note: Read section on the stability and purity of radiolabeled arginine before proceeding

          Incubations of the citrulline assay reaction may be carried out for 10-60 min at 22-37°C, depending on the tissue being used. High levels of nNOS in nervous tissues and skeletal muscle permit brief assay periods (10-15 min) at room temperature. Lower levels of eNOS in vascular tissues require that assays be performed for prolonged periods (60 min).

          Endothelial NOS and nNOS require calcium for enzyme activity; therefore, it is essential to add calcium to the assay medium. A final free calcium concentration of 750 µM is required for optimal NOS activity. When testing NOS activity from tissue extracts, addition of calmodulin to the reaction is not required. However, when testing purified NOS, the addition of calmodulin may be required depending on the type of NOS in question. Nitric oxide synthase activity in the citrulline assay is defined as counts per min (cpm) in an incubated test sample as compared to an appropriate blank. The following control reactions can serve as a blank: a reaction that includes 1 mM NG-Nitro-L-arginine methyl ester HCl (a competitive NOS inhibitor provided at a concentration of 10 mM), a reaction in which the extract is boiled prior to the assay, a reaction in which either NADPH or calcium is omitted or a reaction that is incubated on ice. As in any quantitative enzyme assay, it is important to optimize reaction conditions so that the assay is linear with respect to time and tissue concentration. The NOS in the rat cerebellum extract provided in this kit is linear for at least a 30-min reaction. Specific activity and substrate affinity of NOS can be assessed by carrying out replicate reactions in the presence of varying amounts of unlabeled arginine. The Km (Michaelis constant) of NOS is in the range of 2-20 µM. Appropriate concentrations of arginine for kinetic studies are 0.1-100 µM.

          1. Prepare a reaction stock mix by adding the following components and store on ice.

          Table 2: Stock Mix

          The volumes given here yield sufficient reaction mix for 10 reactions. The reaction mix can be stored on ice for up to 24 h.


          2. Add 40 µl of reaction mix to each microcentrifuge tube.
          3. For the negative controls, add 5 µl of the NOS inhibitor, NG-Nitro-L-arginine methyl ester, HCl, to each tube.
          4. Add 1-10 µl of tissue extract to each tube containing reaction mix. For the control reaction, use 5 µl of the rat cerebellum extract provided. If you are assaying purified eNOS or nNOS, the addition of calmodulin to a final concentration of 0.1 µM is required.

          Note: The rat cerebellum extract should be thawed just before use and should be kept on ice. Do not refreeze the thawed extract.
          5. Incubate the reaction sample at 22-37°C for 10 to 60 min. (For initial experiments, the reactions should be carried out at room temperature for 30 min.)
          6. Stop the reaction by adding 400 µl of stop buffer to the reaction sample.
          7. Thoroughly resuspend the equilibrated resin. Pipet 100 µl of the equilibrated resin into each reaction sample.
          8. Transfer the samples to spin cups and place the spin cups into cup holders.
          9. Centrifuge the spin cups and holders in a microcentrifuge at full speed for 30 s.
          10. Remove the spin cups from the cup holders and transfer the eluate (i.e. the "flowthrough") to scintillation vials. Add scintillation fluid and quantify the radioactivity in a liquid scintillation counter.

          If determining the ratio of unreacted arginine to citrulline

          1. Place the spin cups in fresh microcentrifuge tubes and add 400 µl of elution buffer to spin cup.
          2. Spin the microcentrifuge tubes (with the spin cups in them) in a microcentrifuge at full speed for 30 s.
          3. Remove the spin cup and transfer the eluate to scintillation vials. Add scintillation fluid to the vials and quantify the radioactivity in a scintillation counter.
          4. This value for the quantity of unreacted arginine can be compared with the value for the citrulline produced that was measured in step 10 above.
          Sensitivitypicomolar range
          Assay Range2 h
          Protocol SummaryPreparation of Extracts from Tissues and Cultured Cells Extraction of Proteins from Tissues Add 20 volumes of ice-cold 1X Homogenization Buffer to tissue sample. Homogenize the tissue and spin the homogenate in a microcentrifuge for 5 min at 4°C. Transfer the supernatant to a fresh tube and keep the tube on ice until use. Extraction of Proteins from Tissue Culture Cells Remove the culture media from the tissue culture cells, wash the cells with PBS and harvest the cells in PBS containing 1 mM EDTA Remove the supernatant, and resuspend the cells in 1X Homogenization Buffer. Spin the cells at full speed for 5 min, remove the supernatant and adjust the protein concentration of the sample to 5-10 µg/µl. Measurement of Nitric Oxide Synthase Activity Prepare a reaction mixture in a microcentrifuge tube and place on ice. Add 1-10 µl of tissue extract to 40 µl of the reaction mixture. Incubate the reaction at 22-37°C for 10 to 60 min. Add 400 µl of stop buffer and 100 µl of equilibrated resin to the reaction sample. Spin samples in microfuge at full speed 30 s (using spin cups/holders) Transfer the eluate to a scintillation vial, add scintillation fluid and quantify the radioactivity in a liquid scintillation counter. Measure unreacted arginine if desired to determine ratio of unreacted arginine to citrulline.
          Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
          Poly Prep® is a registered trademark of Bio-Rad Laboratories, Inc.
          Interactive Pathways™ is a trademark of EMD Chemicals, Inc.