eNOS activities were measured by the rate of conversion of [3H] L-arginine to [3H]L-citrulline by using a NOS activity assay kit (Cayman), as per the supplier’s instructions. This assay was used to measure the effects of the test substances on eNOS activity, using recombinant bovine eNOS (Cayman).
Nos activity assay kit
Manufactured by Cayman Chemical
Sourced in United States
The NOS activity assay kit is a laboratory instrument designed to measure the enzymatic activity of nitric oxide synthase (NOS) in biological samples. It provides a quantitative assessment of NOS activity without making inferences about its intended use.
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8 protocols using nos activity assay kit
1
Endothelial NOS Activity Assay
2
Biochemical Assay for nNOS Activity
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The NOS activity assay is based on the biochemical conversion of [3H] l -arginine to [3H] l -citrulline by NOS. Briefly, hearts were homogenized, and the homogenates were centrifuged in a microcentrifuge for 5 min at 4 °C. The supernatant was separated, and the resulting protein concentration was adjusted to 5–10 mg/ml. nNOS activity was measured using a Cayman Chemicals NOS activity assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
3
NOS Activity Assay in PC12 Cells
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Activation of NOS activity by NMDA in PC12 cell transfectants was assessed using the NOS Activity Assay Kit (Cayman Chemical) according to the manufacturer’s instructions. Specifically, 10 μl of proteins extracted from each PC12 cell transfectant sample were incubated with 25 μl of 2X Reaction Buffer, 5 μl of 10 mM NADPH freshly prepared in 10 mM Tris-HCl (pH 7.4), 1 μl of L-[2,3,4,5-3H]arginine monohydrochloride (1 μCi/μl) (Amersham), 5 μl of 6 mM CaCl2 and 4 μl of H2O at room temperature for 60 min. The reaction was stopped by adding 400 μl of Stop Buffer and 100 μl of equilibrated resin. The reaction sample was transferred to a spin cup and centrifuged for 30 sec. The eluate was transferred to a scintillation vial and the radioactivity was quantitated using a liquid scintillation counter.
4
Measuring NOS Activity via Radiolabeled Arginine
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Total NOS activity was assessed by conversion of radiolabeled arginine to citruline ([3H]citruline), as previously described [37 (link)], using a NOS Activity Assay Kit (Cayman Chemical Company, Ann Harbor, MI, USA). Protein content was determined by the Bradford method [38 (link)].
5
Measuring Nitric Oxide Synthase Activity
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NOS activity was determined using an NOS activity assay Kit (Cayman Chemical, Ann Arbor, MI, USA, #781001). Isolated prefrontal cortex tissue of rat brains was homogenized (1/5; w/v) in Assay Buffer, centrifuged (10,000× g, 15 min, 4 °C). The supernatant was used for NOS dependent conversion of 14C L-arginine to 14C L-citrulline, according to manufacturer’s protocol. To distinguish the activity of the iNOS (calcium-independent) and constitutive NOS (nNOS and eNOS), the calcium was chelated by EDTA. The radioactivity was measured in a Wallac 1409 Liquid Scintillation Counter (Perkin–Elmer, Waltharm, MA, USA). The NOS activity was expressed as CPM (counts per minute) of radiolabeled L-citrulline per gram of tissue per min.
6
Quantitative Measurement of eNOS Activity
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eNOS activity was assayed using Cayman’s NOS activity assay kit (catalog No. 781001) following manufacturer’s instruction. Briefly, after appropriate treatments, HCAECs were washed with PBS containing 1 mmol/L EDTA and lysed in homogenization buffer (25 mmol/L Tris-HCl pH 7.4, 1 mmol/L EDTA, and 10 mmol/L EGTA) by brief sonication. eNOS activity was assayed using 20 μg protein from the cell lysate in 50 μL reaction buffer (25 mmol/L Tris-HCl pH 7.4, 3 μmol/L tetrahydrobiopterin, 1 μmol/L flavin adenine dinucleotide, and 1 μmol/L flavin adenine mononucleotide) containing 1 mmol/L NADPH, 1 μCi [3H]-arginine, 0.6 mmol/L CaCl2 and 0.1 μmol/L calmodulin at 37 °C for 20 minutes. In some cases, 1 μmol/L L-NNA was used to inhibit eNOS activity. At the end of incubation period, the assay was stopped by adding 400 μmol/L stop buffer (50 mmol/L HEPES pH 5.5, 2 mmol/L EDTA). Unused arginine was removed by the resin supplied by the manufacturer, and L-citrulline formed during the assay eNOS was quantitated from the eluate using a liquid scintillation counter and the specific activity was calculated.
7
Colorimetric Nitrate Assay for NOS
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A NOS activity assay kit was used to measure newly synthesised NO from l -arginine by the action of NOS in the presence of essential cofactors, according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI, USA). The final products of the reaction were nitrates, measured by a colorimetric method (540 nm), which indirectly represented the activity of NOS. Nitrate concentrations were determined by extrapolating from a standard curve.
8
Myocardial NOS Activity Quantification
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This was performed using the radio immunoassay method. The preparation of the myocardial tissue samples (200 mg) were identical as described previously (Kisvári et al. 2014) (link). Total protein concentrations were determined by the method of Bradford.
The measurements were carried out in the absence of calcium and calmodulin using NOS activity assay kit (Cayman Chemical, Ann Arbor, MI, USA), based on the conversion of [ 3 H] L-arginine to [ 3 H] L-citrulline by NOS. To eliminate background NOS activity in control samples, 10 mmol L-N G -Nitro-arginine (L-NNA), a non-specific inhibitor of the NOS enzyme was given to the reaction mixture before the addition of the tissue extract. A liquid scintillation counter (Wizard TM , PerkinElmer, Waltham, MA, USA) was used to detect the amount of radiolabeled L-citrulline formed during the reaction by iNOS, and expressed as the percentage of the total counts corrected with the background counts per minute.
The measurements were carried out in the absence of calcium and calmodulin using NOS activity assay kit (Cayman Chemical, Ann Arbor, MI, USA), based on the conversion of [ 3 H] L-arginine to [ 3 H] L-citrulline by NOS. To eliminate background NOS activity in control samples, 10 mmol L-N G -Nitro-arginine (L-NNA), a non-specific inhibitor of the NOS enzyme was given to the reaction mixture before the addition of the tissue extract. A liquid scintillation counter (Wizard TM , PerkinElmer, Waltham, MA, USA) was used to detect the amount of radiolabeled L-citrulline formed during the reaction by iNOS, and expressed as the percentage of the total counts corrected with the background counts per minute.
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