Human eNOS/Nitric oxide synthase, endothelial ELISA Kit (HUFI00401)
- SKU:
- HUFI00401
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P29474
- Sensitivity:
- 37.5pg/ml
- Range:
- 62.5-4000pg/ml
- ELISA Type:
- Sandwich
- Synonyms:
- NOS3, eNOS, Constitutive NOS, ECNOS, endothelial, Endothelial NOS, eNOS, cNOS, nitric oxide synthase 3, endothelial cell, NOS type III, NOSIII
- Reactivity:
- Human
- Research Area:
- Cell Biology
Description
Human eNOS/Nitric oxide synthase, endothelial ELISA Kit
The Human eNOS (Nitric Oxide Synthase) Endothelial ELISA Kit is specifically designed for the accurate measurement of eNOS levels in human serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit ensures reliable and reproducible results, providing valuable insights into endothelial function and nitric oxide production in various research applications.Endothelial nitric oxide synthase (eNOS) is a critical enzyme responsible for the production of nitric oxide, a key signaling molecule involved in the regulation of blood pressure, vascular tone, and overall cardiovascular health.
Dysregulation of eNOS activity has been implicated in numerous cardiovascular diseases, making it a crucial biomarker for studying endothelial dysfunction and developing potential therapeutic interventions.By using the Human eNOS Endothelial ELISA Kit, researchers can accurately assess eNOS levels in biological samples, leading to a better understanding of the role of nitric oxide in various physiological and pathological processes. This kit is a valuable tool for advancing cardiovascular research and exploring novel treatment strategies for cardiovascular disorders.
| Product Name: | Human eNOS / Nitric oxide synthase, endothelial ELISA Kit |
| Product Code: | HUFI00401 |
| Size: | 96 Assays |
| Alias: | NOS3, eNOS, Constitutive NOS, ECNOS, endothelial, Endothelial NOS, eNOS, cNOS, nitric oxide synthase 3, endothelial cell, NOS type III, NOSIII |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human NOS3/eNOS concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 37.5pg/ml |
| Range: | 62.5-4000pg/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human NOS3/eNOS and the recovery rates were calculated by comparing the measured value to the expected amount of Human NOS3/eNOS in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NOS3/eNOS and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
| Uniprot | P29474 |
| UniProt Protein Function: | eNOS: endothelial constitutive nitric oxide synthase. Synthesizes nitric oxide (NO) from arginine and oxygen, which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets. Induced in humans by caloric restriction. Stimulated by calcium/calmodulin. Inhibited by NOSIP and NOSTRIN. |
| UniProt Protein Details: | Protein type:Amino Acid Metabolism - arginine and proline; Oxidoreductase; EC 1.14.13.39 Chromosomal Location of Human Ortholog: 7q36 Cellular Component: caveola; cytoplasm; cytoskeleton; cytosol; Golgi membrane; nucleus; plasma membrane Molecular Function:actin monomer binding; cadmium ion binding; calmodulin binding; FAD binding; FMN binding; heme binding; iron ion binding; NADP binding; nitric-oxide synthase activity; protein binding Biological Process: angiogenesis; arginine catabolic process; blood coagulation; blood vessel remodeling; cell redox homeostasis; endothelial cell migration; in utero embryonic development; lipopolysaccharide-mediated signaling pathway; lung development; mitochondrion organization and biogenesis; negative regulation of blood pressure; negative regulation of calcium ion transport; negative regulation of cell proliferation; negative regulation of hydrolase activity; negative regulation of muscle hyperplasia; negative regulation of potassium ion transport; nitric oxide biosynthetic process; nitric oxide mediated signal transduction; nitric oxide metabolic process; ovulation from ovarian follicle; positive regulation of angiogenesis; positive regulation of guanylate cyclase activity; positive regulation of vasodilation; regulation of blood pressure; regulation of blood vessel size; regulation of nitric-oxide synthase activity; regulation of sodium ion transport; regulation of systemic arterial blood pressure by endothelin; regulation of the force of heart contraction by chemical signal; removal of superoxide radicals; response to heat; smooth muscle hyperplasia; vascular endothelial growth factor receptor signaling pathway Disease: Alzheimer Disease; Hypertension, Essential; Preeclampsia/eclampsia 1; Stroke, Ischemic |
| NCBI Summary: | Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2009] |
| UniProt Code: | P29474 |
| NCBI GenInfo Identifier: | 266648 |
| NCBI Gene ID: | 4846 |
| NCBI Accession: | P29474.3 |
| UniProt Secondary Accession: | P29474,Q13662, Q14251, Q14434, Q548C1, Q6GSL5, Q9UDC6 A0S0A7, A0S0A8, A8KA63, B2RCQ1, E9PFR2, |
| UniProt Related Accession: | P29474 |
| Molecular Weight: | |
| NCBI Full Name: | Nitric oxide synthase, endothelial |
| NCBI Synonym Full Names: | nitric oxide synthase 3 |
| NCBI Official Symbol: | NOS3Â Â |
| NCBI Official Synonym Symbols: | eNOS; ECNOSÂ Â |
| NCBI Protein Information: | nitric oxide synthase, endothelial |
| UniProt Protein Name: | Nitric oxide synthase, endothelial |
| UniProt Synonym Protein Names: | Constitutive NOS; cNOS; EC-NOS; Endothelial NOS; eNOS; NOS type III; NOSIII |
| Protein Family: | Nitric oxide synthase |
| UniProt Gene Name: | NOS3Â Â |
| UniProt Entry Name: | NOS3_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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