Nitrotyrosine

Manufactured by Santa Cruz Biotechnology

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Nitrotyrosine is a chemical compound that is used to detect and quantify the levels of nitrotyrosine in biological samples. Nitrotyrosine is formed when tyrosine residues in proteins are modified by reactive nitrogen species, such as peroxynitrite, and is considered a marker of nitrosative stress.

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Anti-nitrotyrosine (12 citations) 3-nitrotyrosine (3 citations) Anti-3-nitrotyrosine (2 citations) Rabbit anti-nitrotyrosine (2 citations) Anti-nitrotyrosine (NT; (2 citations) Rabbit anti-p-c-fms/CSF-1R(Tyr723) and nitrotyrosine (2 citations)

21 protocols using nitrotyrosine

Immunostaining of Nitrotyrosine in Paraffin Samples

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Paraffin-embedded samples were prepared according to standard histological procedures. Nitrotyrosine (1:200 dilution; Santa Cruz, CA, United States) was used for immunostaining. The sections were then incubated with a goat anti-Mouse IgG superclonal secondary antibody for 1 h. Fluorescence images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics).

Yang Y., Zhao M., He X., Wu Q., Li D.L, & Zang W.J. (2021). Pyridostigmine Protects Against Diabetic Cardiomyopathy by Regulating Vagal Activity, Gut Microbiota, and Branched-Chain Amino Acid Catabolism in Diabetic Mice. Frontiers in Pharmacology, 12, 647481.

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Immunohistochemical Analysis of Adipose Tissue

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A commercially available kit based on immunoperoxidase was used for immunohistochemistry (Dako, Glostrup, Denmark). Briefly, epididymal adipose tissues from WT and CKO mice on ND or HFD were fixed in 10% formalin, dehydrated, embedded in paraffin wax, and subsequently deparaffinized. Endogenous peroxidase was quenched for 30 min using Dako peroxidase solution, and tissue sections were then washed and blocked with Dako serum-free blocking solution. Sections were subsequently probed overnight at 4°C with antibodies against F4/80 (1 : 100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), CD11c (1 : 200, Santa Cruz Biotechnology Inc.), CD206 (1 : 50, Santa Cruz Biotechnology Inc.), and nitrotyrosine (1 : 200, Santa Cruz Biotechnology Inc.). After washing in phosphate-buffered saline, sections were processed with a LSAB2 Kit (Dako) and labeled with 3,3′-diaminobenzidine for 4 min. Digital images were captured on a Zeiss microscope equipped with an Axio Cam HRC digital camera and software (Carl Zeiss, Thornwood, NY, USA) and analyzed with the open-source program ImageJ v1.34s (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA).

Park Y.S., Uddin M.J., Piao L., Hwang I., Lee J.H, & Ha H. (2016). Novel Role of Endogenous Catalase in Macrophage Polarization in Adipose Tissue. Mediators of Inflammation, 2016, 8675905.

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Protein Extraction and Antibody Detection in Myocardial Tissues

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The preparation of protein samples from myocardial tissues was performed as previously described (Becker et al. 2005 (link)). Antibodies to eNOS (BD Transduction Laboratories, 1:250 dilution), phospho-eNOS (Ser 1177) (Cell Signal, 1:500 dilution), SOD-1(Calbiochem, 1:5000 dilution), SOD-2 (BD Transduction Laboratories, 1:10,000 dilution), SOD-3 (Santa Cruz Biotechnology, 1:5000 dilution), or one of the following subunits of NAD(P)H oxidase: p67^phox (Upstate, 1:1000 dilution), p22^phox (Santa Cruz Biotechnology, 1:2000 dilution), gp91^phox(BD Transduction Laboratories, 1:1000 dilution), p47^phox (Santa Cruz Biotechnology, 1:1000 dilution), phos-p47^phox (Upstate, 1:1000 dilution), p40^phox (Santa Cruz Biotechnology, 1:800 dilution), Rac-1 (Santa Cruz Biotechnology, 1:5000 dilution), and nitrotyrosine (Santa Cruz Biotechnology, 1:1000 dilution) were used.

Song S., Kertowidjojo E., Ojaimi C., Martin-Fernandez B., Kandhi S., Wolin M, & Hintze T.H. (2015). Long-term methionine-diet induced mild hyperhomocysteinemia associated cardiac metabolic dysfunction in multiparous rats. Physiological Reports, 3(5), e12292.

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Fibroblast Protein Expression Analysis

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Fibroblast levels of relevant proteins were assessed by Western blot as previously described (39 (link)). Briefly, fibroblasts were lysed and homogenized with lysis buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na₃VO₄, 10 mg/mL leupeptin, 10 mg/mL aprotinin, and 20 mM PMSF]. Proteins from each sample were separated on 10–15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin (Wisent Inc, St-Bruno, QC, Canada), the membranes were probed with primary antibodies against mouse NLRP3, caspase 1 p20 (Adipogen, San Diego, CA, USA) at 1:500 dilution; pro-IL-1β (R and D Systems, Minneapolis, MN, USA) at 1:500 dilution; nitrotyrosine (Santa Cruz, Dallas, TX, USA) at 1:1,000 dilution; and β-actin (Santa Cruz, Dallas, TX, USA) at 1:2,000 dilution. After treatment with appropriate secondary antibodies, the specific bands were detected with an enhanced chemiluminescence (ECL) system and quantified with an imaging densitometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Lan T., Tao A., Xu X., Kvietys P, & Rui T. (2020). Peroxynitrite/PKR Axis Modulates the NLRP3 Inflammasome of Cardiac Fibroblasts. Frontiers in Immunology, 11, 558712.

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Protein Expression Analysis by Western Blot

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Proteins were electrophoresed on SDS polyacrylamide gels, transferred to Hybond-c Extra nitrocellulose membranes (Bio-Rad) and incubated with primary antibodies for: AKAP-12 (Santa Cruz Biotechnology; 1:100), Nitrotyrosine (Santa Cruz Biotechnology; 1:100), CML (Abcam; 1:200), MDA (Abcam; 1:200), PGC1-α (Santa Cruz Biotechnology; 1:100), PHB (Cell Signaling; 1:100). After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Bio-Rad). Results are expressed as an n-fold increase over the values of the control group in densitometric arbitrary units. All Western Blots were performed at least in triplicate for each experimental condition.

Ibarrola J., Sadaba R., Martinez-Martinez E., Garcia-Peña A., Arrieta V., Alvarez V., Fernández-Celis A., Gainza A., Cachofeiro V., Santamaria E., Fernandez-Irigoyen J., Jaisser F, & Lopez-Andres N. (2018). Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12. Scientific Reports, 8, 6801.

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Cardiac Tissue Immunohistochemistry Analysis

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Histological determinations in cardiac tissue were performed in 5 μm-thick sections. Slides were treated with H₂O₂ and incubated with AKAP-12 (Santa Cruz; 1:100), Nitrotyrosine (Santa Cruz; 1:5000), CML (Abcam; 1:2000), PGC-1α (Santa Cruz; 1:100) washed three times, and then incubated with the horseradish peroxidase-labeled polymer conjugated to secondary antibodies (Dako Cytomation). The signal was revealed by using DAB Substrate Kit (BD Pharmingen). As negative controls, samples followed the same procedure described above but in the absence of primary antibodies. All measurements and quantifications were performed blind in an automated image analysis system (Nikon).

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Protein Expression Profiling of Neurodegenerative Markers

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Striatal tissue lysates (50 μg protein) and SH-SY5Y cell lysates were separated by SDS-PAGE on 10–12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with specific primary antibodies and the antibodies used were DJ-1, Nrf2, phosphoS40-Nrf2 (Abcam 1 : 1000 dilution), Keap1, NQO1, Cullin3, PKC-δ (Pierce Antibodies, 1 : 1000 dilution), γGCLC, HO-1, nitrotyrosine (Santa Cruz Biotech, 1 : 1000 dilution), α-synuclein, and iNOS (Cell Signaling Technology, 1 : 1000 dilution). To verify the uniformity of protein load and transfer efficiency across the test samples, membranes were reprobed with β-actin and Lamin B (Cell Signaling Technology, 1 : 1000 dilution). Immunoreactive bands were developed with Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA) and visualized by using an enhanced chemiluminescence system (ChemiDoc, Bio-Rad, USA) and presented in comparison to β-actin/Lamin B expression.

Narasimhan K.K., Jayakumar D., Velusamy P., Srinivasan A., Mohan T., Ravi D.B., Uthamaraman S., Sathyamoorthy Y.K., Rajasekaran N.S, & Periandavan K. (2019). Morinda citrifolia and Its Active Principle Scopoletin Mitigate Protein Aggregation and Neuronal Apoptosis through Augmenting the DJ-1/Nrf2/ARE Signaling Pathway. Oxidative Medicine and Cellular Longevity, 2019, 2761041.

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Western Blot Analysis of Nerve Tissue

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For Western blot analyses, the tail nerve, DRG and spinal cord tissue was homogenized in 50 (nerve) or 100 μl (DRG and spinal cord) lysis buffer (10 mM Tris base, 2 mM EDTA, 150 mM sodium chloride, 1% Triton-X 100, and 1 μl/ml Sigma Protease inhibitor cocktail^™), and protein concentrations were measured using Bio-Rad _DC assay (Bio-Rad Inc.). The homogenates were diluted with Laemmli buffer (1:1.5), separated on a 10% SDS–PAGE gel, and transferred to nitrocellulose membranes. The membranes were blocked in LI-COR blocking buffer (LI-COR Biosciences, Lincoln NE) and probed for nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA) or Beta-actin (Cell Signaling Technologies, Danvers, MA). The detection was performed with IRDye800-conjugated anti-mouse, or anti-rabbit IgG, respectively in LI-COR blocking buffer. At the end of incubation period, the membranes were washed 3× with TBST. The membranes were visualized and analyzed on Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE). Band signal intensities are presented as area intensity relative to control samples after normalization to beta actin as loading control.

Pacurari M., Waugh S, & Krajnak K. (2018). Acute Vibration Induces Peripheral Nerve Sensitization in a Rat Tail Model: Possible Role of Oxidative Stress and Inflammation. Neuroscience, 398, 263-272.

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Antibody-Based Analysis of Neuronal Stress

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Mouse monoclonal antibodies against tyrosine phospho-Hydroxylase (Ser31), GAPDH, β-actin, 4-Hydroxy-2-nonenal (4-HNE), Nitro-tyrosine, CHOP, Fas, Bax, NFkB p65, Histone H1, TRAF2, IRE1α, and phospho-IKB-β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit monoclonal antibodies against phospho-p38 MAPK (Thr180/Tyr182) and phospho-JNK1/2 (Thr183/Tyr185) were purchased from Cell Signaling Technology (Beverly, MA, USA). The TdT-mediated dUTP Nick End Labeling (TUNEL) kits were purchased from Roche (Germany). MPP+ (1-methyl-4-phenylpyridinium), SDS, NP-40, while sodium deoxycholate, protease inhibitor cocktail was purchased from Sigma (St. Louis, MO, USA).

Kuo H.C., Lu C.C., Shen C.H., Tung S.Y., Hsieh M.C., Lee K.C., Lee L.Y., Chen C.C., Teng C.C., Huang W.S., Chen T.C, & Lee K.F. (2016). Hericium erinaceus mycelium and its isolated erinacine A protection from MPTP-induced neurotoxicity through the ER stress, triggering an apoptosis cascade. Journal of Translational Medicine, 14, 78.

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Histological Analysis of Joint Inflammation

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Mouse joint tissue was fixed in 4% paraformaldehyde, decalcification EDTA bone decalcifier and embedded in paraffin. The section (7 μm) was stained with hematoxylin and eosin, Safranin O, and toluidine blue to detect proteoglycans.
Immunohistochemistry was performed using the Vectastatin ABC kit (Vector Laboratories, Burlingame, CA, USA). Tissue was first incubated with primary antibodies to IL-17, IL-6, IL-1b, TNF-α, Nitrotyrosine, NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HO-1, Inos (Abcam, Cambridge Science Park, Cambridge, UK) overnight at 4°C. The sections were counterstained with hematoxylin. Samples were photographed with an Olympus photomicroscope (Tokyo, Japan).

Jhun J., Lee J., Byun J.K., Kim E.K., Woo J.W., Lee J.H., Kwok S.K., Ju J.H., Park K.S., Kim H.Y., Park S.H, & Cho M.L. (2014). Red Ginseng Extract Ameliorates Autoimmune Arthritis via Regulation of STAT3 Pathway, Th17/Treg Balance, and Osteoclastogenesis in Mice and Human. Mediators of Inflammation, 2014, 351856.

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