Nitrotyrosine ELISA Kit

Produktnummer ABIN2344944

Produktdetails Nitrotyrosine ELISA Kit

Target: Nitrotyrosine

Reaktivität: Chemical

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Applikation: ELISA

Verwendungszweck: The nitrotyrosine quantitation kit is a competitive ELISA.

Marke: OxiSelect™

Proben: Plasma, Serum

Analytische Methode: Quantitative

Sensitivität: 20 nM

Produktmerkmale: The kit has a nitrotyrosine detection sensitivity range of 20 nM to 8.0 μM. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.

Bestandteile:

1. Nitrotyrosine Coated EIA Plate : one strip well 96-well plate.
2. Anti-Nitrotyrosine Antibody : One 20 μL vial of anti-nitrotyrosine Rabbit IgG.
3. Secondary Antibody, HRP Conjugate : One 20 μL vial.
4. Assay Diluent : One 50 mL bottle.
5. 10X Wash Buffer : One 100 mL bottle.
6. Substrate Solution : One 12 mL amber bottle.
7. Stop Solution (Part. No. 310808): One 12 mL bottle.
8. Nitrated BSA Standard : One 500 μL vial of 1 mg/mL Nitrated BSA in PBS with a nitrotyrosine content of 40 μM (2.7 mole of nitrotyrosine per mole of BSA). The protein nitrotyrosine level is predetermined by a spectrophotometric method as described by Ischiropoulos et al (See Ref. 3).

Benötigtes Material:

1. Protein samples such as purified protein, plasma, serum, cell lysate
2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
4. Multichannel micropipette reservoir
5. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Sensitive detection of 3-Nitrotyrosine as low as 10 nM
• Suitable for use with cell lysates, serum, plasma and purified proteins
• Nitrated BSA provided as positive control

Plattentyp: Pre-coated

Protokoll: The unknown protein nitrotyrosine sample or nitrated BSA standards are first added to a nitrated BSA preabsorbed EIA plate. After a brief incubation, an anti-nitrotyrosine antibody is added, followed by an HRP conjugated secondary antibody. The protein nitrotyrosine content in unknown sample is determined by comparing with a standard curve that is prepared from predetermined nitrated BSA standards.

Aufbereitung der Reagenzien:

• 1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir to homogeneity. 3
• Anti-Nitrotyrosine Antibody and Secondary Antibody: Immediately before use dilute the Anti- Nitrotyrosine Antibody 1:1000 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Testdurchführung:

1. Prepare and mix all reagents thoroughly before use. Each protein sample including nitrated BSA and blank should be assayed in duplicate.
2. Add 50 μL of unknown protein sample or nitrated BSA standard to the wells of the EIA plate. Incubate at room temperature for 10 minutes on an orbital shaker.
3. Add 50 μL of the diluted anti-nitrotyrosine antibody to each well, incubate at room temperature for 1 hour on an orbital shaker.
4. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
5. Add 100 μL of the diluted Secondary Antibody-Enzyme Conjugate to all wells.
6. Incubate at room temperature for 1 hour on an orbital shaker.
7. Wash microwell strips 3 times according to step 4 above. Proceed immediately to the next step.
8. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation. 4
9. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
10. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid multiple freeze/thaw cycles.

Lagerung: 4 °C/-20 °C

Informationen zur Lagerung: Upon receipt, aliquot and store the Nitrated BSA Standard at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C until their expiration dates.

Referenzen für Nitrotyrosine ELISA Kit (ABIN2344944)

Shafique, Torina, Reichert, Colantuono, Nur, Zeeshan, Ravichandran, Liu, Feng, Zeeshan, Benjamin, Irani, Harrington, Sellke, Abid: "Mitochondrial redox plays a critical role in the paradoxical effects of NAPDH oxidase-derived ROS on coronary endothelium." in: Cardiovascular research, Vol. 113, Issue 2, pp. 234-246, (2017) (PubMed).

Lambrechts, Balestra, Theron, Henckes, Galinat, Mignant, Belhomme, Pontier, Guerrero: "Venous gas emboli are involved in post-dive macro, but not microvascular dysfunction." in: European journal of applied physiology, Vol. 117, Issue 2, pp. 335-344, (2017) (PubMed).

Wu, Li, Zhu, Wang, Dai, Zhang, Zheng, Xu, Wang, Zhang, Zhou, Zhang, Shi: "Mdivi-1 Alleviates Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats, Possibly via Inhibition of Drp1-Activated Mitochondrial Fission and Oxidative Stress." in: Neurochemical research, Vol. 42, Issue 5, pp. 1449-1458, (2017) (PubMed).

Toth, Tarantini, Ashpole, Tucsek, Milne, Valcarcel-Ares, Menyhart, Farkas, Sonntag, Csiszar, Ungvari: "IGF-1 deficiency impairs neurovascular coupling in mice: implications for cerebromicrovascular aging." in: Aging cell, Vol. 14, Issue 6, pp. 1034-44, (2016) (PubMed).

Lux, Pokreisz, Swinnen, Caluwe, Gillijns, Szelid, Merkely, Janssens: "Concomitant Phosphodiesterase 5 Inhibition Enhances Myocardial Protection by Inhaled Nitric Oxide in Ischemia-Reperfusion Injury." in: The Journal of pharmacology and experimental therapeutics, Vol. 356, Issue 2, pp. 284-92, (2016) (PubMed).

Sataranatarajan, Qaisar, Davis, Sakellariou, Vasilaki, Zhang, Liu, Bhaskaran, McArdle, Jackson, Brooks, Richardson, Van Remmen: "Neuron specific reduction in CuZnSOD is not sufficient to initiate a full sarcopenia phenotype." in: Redox biology, Vol. 5, pp. 140-8, (2016) (PubMed).

Sumayao, McEvoy, Newsholme, McMorrow: "Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox-sensitive genes in human kidney proximal tubular cells." in: The Journal of physiology, (2016) (PubMed).

Adhami, Starck, Flores, Martins Green: "A Health Threat to Bystanders Living in the Homes of Smokers: How Smoke Toxins Deposited on Surfaces Can Cause Insulin Resistance." in: PLoS ONE, Vol. 11, Issue 3, pp. e0149510, (2016) (PubMed).

Paul, Arya, Gangwar, Bhargava, Ahmad: "Size restricted silymarin suspension evokes integrated adaptive response against acute hypoxia exposure in rat lung." in: Free radical biology & medicine, Vol. 96, pp. 139-51, (2016) (PubMed).

Dhall, Alamat, Castro, Sarker, Mao, Chan, Hang, Martins-Green: "Tobacco toxins deposited on surfaces (third hand smoke) impair wound healing." in: Clinical science (London, England : 1979), Vol. 130, Issue 14, pp. 1269-84, (2016) (PubMed).

Chan, Loh, Peh, Tan, Tan, Li, Tay, Wong, Engelward: "House dust mite-induced asthma causes oxidative damage and DNA double-strand breaks in the lungs." in: The Journal of allergy and clinical immunology, Vol. 138, Issue 1, pp. 84-96.e1, (2016) (PubMed).

Saenen, Vrijens, Janssen, Roels, Neven, Vanden Berghe, Gyselaers, Vanpoucke, Lefebvre, De Boever, Nawrot: "Lower Placental Leptin Promoter Methylation in Association with Fine Particulate Matter Air Pollution during Pregnancy and Placental Nitrosative Stress at Birth in the ENVIRONAGE Cohort." in: Environmental health perspectives, Vol. 125, Issue 2, pp. 262-268, (2016) (PubMed).

Liu, Zhao, Yue, Wang, Li, Guo, Ma, Yao, Gao, Deng, Li, Feng, Qu: "Pterostilbene Attenuates Early Brain Injury Following Subarachnoid Hemorrhage via Inhibition of the NLRP3 Inflammasome and Nox2-Related Oxidative Stress." in: Molecular neurobiology, (2016) (PubMed).

Ciarlone, Dean: "Acute hypercapnic hyperoxia stimulates reactive species production in the caudal solitary complex of rat brain slices but does not induce oxidative stress." in: American journal of physiology. Cell physiology, Vol. 311, Issue 6, pp. C1027-C1039, (2016) (PubMed).

Bavunoglu, Genc, Konukoglu, Cicekci, Sozer, Gelisgen, Uzun: "Oxidative stress parameters and inflammatory and immune mediators as markers of the severity of sepsis." in: Journal of infection in developing countries, Vol. 10, Issue 10, pp. 1045-1052, (2016) (PubMed).

Mikolka, Kopincová, Košútová, Čierny, Čalkovská, Mokrá: "Lung inflammatory and oxidative alterations after exogenous surfactant therapy fortified with budesonide in rabbit model of meconium aspiration syndrome." in: Physiological research, Vol. 65, Issue Supplementum 5, pp. S653-S662, (2016) (PubMed).

Mikolka, Kopincova, Mikusiakova, Kosutova, Calkovska, Mokra: "Antiinflammatory Effect of N-Acetylcysteine Combined with Exogenous Surfactant in Meconium-Induced Lung Injury." in: Advances in experimental medicine and biology, Vol. 934, pp. 63-75, (2016) (PubMed).

Martinez, Bonomo, Guizoni, Junior, Damatto, Cezar, Lima, Pagan, Seiva, Bueno, Fernandes, Laurindo, Zornoff, Okoshi, Okoshi: "Modulation of MAPK and NF-954;B Signaling Pathways by Antioxidant Therapy in Skeletal Muscle of Heart Failure Rats." in: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, Vol. 39, Issue 1, pp. 371-84, (2016) (PubMed).

Kusku-Kiraz, Genc, Bekpinar, Unlucerci, Olgac, Uysal, Gurdol: "Circulating levels of apelin, glucagon-like peptide and visfatin in hypercholesterolemic-hyperhomocysteinemic guinea-pigs: their relation with NO metabolism." in: Molecular and cellular biochemistry, Vol. 400, Issue 1-2, pp. 69-75, (2015) (PubMed).

Chou, Reiter, Chen, Leu, Wang, Yeh: "Pulmonary function changes in rats with taurocholate-induced pancreatitis are attenuated by pretreatment with melatonin." in: Journal of pineal research, Vol. 56, Issue 2, pp. 196-203, (2015) (PubMed).

Details zu Nitrotyrosine

Target: Nitrotyrosine

Abstract: Nitrotyrosine Produkte

Substanzklasse: Chemical

Hintergrund: The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite (Figure 1) or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. Detection of nitrotyrosine-containing proteins has been reported in many human and animal diseases or cellular models of disease. While all tyrosine residues in proteins may theoretically be targets for nitration, presumably the efficiency of tyrosine nitration is dependent on various biological conditions such as the local production and concentration of the reactive species, the existence and availability of antioxidants and scavengers, the accumulation of inflammatory cell and the presence of pro- inflammatory cytokines, as well as the proximity and compartmentation of these components. The quantity of 3-nitrotyrosine in protein sample is determined by comparing its absorbance with that of a known nitrated BSA standard curve.