Antibodies for β-actin (sc-47778), COX2 (sc-19999), MMP1 (sc-21731), NRF1 (sc-101102), and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. Antibodies for acetyl-histone H4 K5, K8, K12, and K16 (H4K5,8,12,16ac, #PA5-40084) were obtained from Invitrogen. Antibodies for anti-histone H3 acetyl K9, K14, K18, K23, K27 (H3K9, 14, 18, 23, 27ac, ab47915), ERα (ab3575), ERβ (ab3576), H4K20me1 (ab9051), H4K20me3 (ab9053), H4R3me1 (ab17339), H3K9me2 (ab1220), H3K9me3 (ab8898), H3K27me2 (ab24684) and H3K27me3 (ab6002), H2AX (ab20669), and γH2AX (ab2893) were obtained from Abcam. The antibody for 8-oxo-dG (4354-MC-050) was obtained from Novus Biologicals. The 3-nitrotyrosine (3-NT) was measured using 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam) per manufacturers’ instructions. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) according to manufacturers’ instructions. The protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology) per manufacturers’ instructions. Luciferase activity assay was carried out using the Dual-Luciferase™ Assay System (Promega) and the transfection efficiency was normalized using a cotransfected renilla plasmid (23 (link)). 17β-estradiol (E2, #E2758) and TNFα (#T0157) were obtained from Sigma.
3 nitrotyrosine elisa kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The 3-Nitrotyrosine ELISA Kit is a quantitative assay designed to measure the levels of 3-nitrotyrosine, a post-translational modification associated with oxidative stress and inflammatory conditions. The kit utilizes an enzyme-linked immunosorbent assay (ELISA) format to detect and quantify the target analyte in biological samples.
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Lab products found in correlation
Coomassie protein assay kit, by Thermo Fisher Scientific (3 mentions)
Vcam1 elisa kit, by Thermo Fisher Scientific (3 mentions)
Ab6002, by Abcam (2 mentions)
Pierce mitochondria isolation kit, by Thermo Fisher Scientific (2 mentions)
Il 6 elisa kit, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrate nitrite colorimetric assay kit, by Cayman Chemical (2 mentions)
Ab3576, by Abcam (1 mentions)
Ab9051, by Abcam (1 mentions)
Ab17339, by Abcam (1 mentions)
Ab24684, by Abcam (1 mentions)
Ab9053, by Abcam (1 mentions)
Sc 30080, by Santa Cruz Biotechnology (1 mentions)
17β estradiol e2 e2758, by Merck Group (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Tnf α elisa kit, by Abcam (1 mentions)
Methylglyoxal assay kit, by Abcam (1 mentions)
Gsh gssg ratio detection assay kit fluorometric green, by Abcam (1 mentions)
Nrf2 transcription factor assay kit colorimetric, by Abcam (1 mentions)
Ab37438, by Abcam (1 mentions)
20 protocols using 3 nitrotyrosine elisa kit
1
Antibody-based Protein Quantification in Cellular Studies
2
Myocardial Oxidative and Inflammatory Markers
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Myocardial levels (n = 8–10 samples) of oxidative stress and inflammatory markers were tested using enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits: 3-nitrotyrosine ELISA kit (ab116691; Abcam), lipid peroxidation (malondialdehyde) assay kit (ab118970; Abcam), interleukin-6 (IL6) ELISA kit (ab100772; Abcam), intercellular cell adhesion molecule-1 (ICAM1) ELISA kit (ERICAM1; Thermo Fisher Scientific), vascular cell adhesion molecule-1 (VCAM1) ELISA kit (KHT0601; Thermo Fisher Scientific), and tumor necrosis factor alpha (TNFα) ELISA kit (ab108913; Abcam). Hydrogen peroxide (H2O2) was assessed in LV tissue homogenates (n = 8–10/group). Samples containing equal amounts of total protein were analyzed for H2O2 formation. H2O2 formation was measured by colorimetry at 540 nm. Results were converted using the standard curve for a known concentration of H2O2.
3
Measuring Protein Oxidative Damage via 3-Nitrotyrosine
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3-Nitrotyrosine modification of proteins is a well-established marker of protein damage by oxidative stress [37 (link)]. Kidney slides were homogenized with an extraction buffer supplemented with protease inhibitor cocktail (Thermo) and protein oxidative damage was measured with the 3-Nitrotyrosine ELISA kit (Abcam) according to the manufacturer's instructions.
4
Nrf2 Activation and Oxidative Stress Markers
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The Glutathione/Glutathione disulfide (GSH/GSSG) ratio was determined using the GSH/GSSG ratio detection assay kit (fluorometric - green) (#ab205811 from Abcam) and the transcriptional activity of Nrf2 was determined using the Nrf2 transcription factor assay kit (colorimetric) (#ab207223 from Abcam). Methylglyoxal (MG) formation was determined using the methylglyoxal assay kit (#ab241006 from Abcam) and 3-nitrotyrosine formation was measured using the 3-nitrotyrosine ELISA kit (#ab116691 from Abcam) according to the manufacturer’s instructions.
5
Quantifying Protein Modifications in Cells
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The antibodies for ERRα (ab37438), H2AX (ab20669) and γH2AX (ab2893), H3K9me2 (ab1220), and H3K9me3 (ab8898) and H3K27me3 (ab6002) were obtained from Abcam. Antibodies for β-actin (sc-47,778), ERβ (sc-137,381), and SOD2 (sc-30,080) were obtained from Santa Cruz Biotechnology. LNG and EE were obtained from Sigma. 3-Nitrotyrosine (3-NT) was measured using the 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam) per manufacturers’ instructions. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) per manufacturers’ instructions. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology). Protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology).
6
Epithelial Cell Culture and Protein Analysis
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Human primary mammalian epithelial cells (HMECs, obtained from Lonza) were cultured in MEGM BulletKit (CC-3150). MCF7 and MDA-MB-231 (MDA231, obtained from ATCC) were cultured in DMEM at 37°C supplemented with 10% FBS and antibiotics. Antibodies for β-actin (sc-47778), Ki-67 (sc-101861), SREBP1 (sc-13551), SREBP2 (sc-13552), and VDAC1 (sc-390996) were obtained from Santa Cruz Biotechnology. Antibodies for HKDC1 (ab228729) and PGC1β (ab176328) were obtained from Abcam. 3-nitrotyrosine (3-NT) was measured using the 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam). The Coomassie Protein Assay Kit (Pierce Biotechnology) was used to measure the protein concentration. The siRNA for SREBP1, SREBP2, PGC1β, and negative control (#AM4636) were purchased from Ambion. The Lipofectamine™ Reagent (Invitrogen) was used for DNA transfection (5 (link)).
7
Quantifying Peroxynitrite via 3-Nitrotyrosine
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Measuring 3-nitrotyrosine (3-NT) indirectly provides an estimate of peroxynitrite in the samples. 3-NT activity was determined using a commercially available 3-nitrotyrosine ELISA kit (Abcam, Cambridge, England), which has the microplates coated with a nitrotyrosine containing antigen. Fifty microliters of standards and samples was added to the designated wells followed by 50 µl horseradish peroxidase-conjugated anti 3-nitrotyrosine antibody. Plate was incubated on a shaker for 2 h at room temperature. The solution in each well was aspirated and washed with 300 µl Tris buffer saline four times. One hundred microliters of H2O2 was added, and the plate was read in the kinetic mode for 15 min at 1 min intervals at absorbance of 600 nm using a microplate reader.
8
Measuring Oxidative Stress Biomarker Nitrotyrosine
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Because nitrotyrosine is a product of protein tyrosine nitration resulting from oxidative damage to proteins by peroxynitrite, we measured the abundance of nitrotyrosine as an oxidative stress marker in muscle. The medial portion of the gastrocnemius muscle was homogenized and total protein was extracted using Cell Extraction Buffer (Invitrogen, Waltham, MA, USA). Total protein concentration was determined as described above. The abundance of nitrotyrosine in the protein samples was measured using a 3-Nitrotyrosine ELISA kit (Abcam, Cambridge, MA, USA) on the DTX800 Multi-mode microplate reader (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instruction.
9
Oxidative Stress Markers in NMR and Mouse
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2.5×105 NMR and mouse BMDM were polarized for 24 h, and then cells were collected and frozen. Content of 3-nitrotyrosine in cell lysates was estimated with help of 3-Nitrotyrosine ELISA Kit (Abcam, USA) according to manufacturer protocol using Thermofisher “Fluoroscan” microplate reader (USA) at 600 nm. Product of lipid peroxidation 4-hydroxynonenal in lysate of macrophages from NMR and mouse was measured using to “Lipid Peroxidation Assay Kit” (Abcam, USA) and Thermofisher “Fluoroscan” microplate reader (USA) (450 nm). Calibration was done with standards provided in the kit and the values expressed as mean of three replicates with standard deviation in pmol/mg of protein.
10
Myocardial Oxidative Stress and Inflammation Assays
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Myocardial levels (n = 4–5 sample/group) of oxidative stress and inflammatory markers were tested with enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits: Hydrogen peroxide assay kit (ab102500; Abcam, Cambridge, UK), lipid peroxidation (malondialdehyde) assay kit (ab118970; Abcam), 3-Nitrotyrosine ELISA kit (ab116691; Abcam), nitric oxide (NO) colorimetric assay kit (K262; BioVision, Inc., Milpitas, CA, USA), interleukin-6 (IL-6) ELISA kit (ab100772; Abcam), intercellular cell adhesion molecule-1 (ICAM-1) ELISA kit (ERICAM1; Thermo Fisher Scientific, Waltham, MA, USA), vascular cell adhesion molecule-1 (VCAM-1) ELISA kit (KHT0601; Thermo Fisher Scientific), tumor necrosis factor alpha (TNFα) ELISA kit (ab108913; Abcam), transforming growth factor-beta 1 (TGF-β1) ELISA kit (BMS623-3; Thermo Fisher Scientific), fibronectin 1 (FN-1) ELISA kit (LS-F2425; LifeSpan BioSciences, Seattle, WA, USA).
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