Cytokine and protein concentrations (interleukin- [IL-] 1β, IL-10, aggrecan [ACAN], basic fibroblast growth factor [bFGF], bone morphogenetic protein- [BMP-] 2 and BMP-7, and collagen type 2 cleavage [C2C]) in synovial joint fluids from acutely infected knee joints or coculture supernatants were analyzed by ELISA (RnD, Minneapolis, MN, USA, and BioSource Deutschland GmbH, Solingen, Germany) according to the manufacturers' instructions. The Nitrotyrosine ELISA Kit was purchased from Abcam (Cambridge, UK). Briefly, this assay employs the quantitative sandwich enzyme immunoassay technique. The microplate was precoated with a specific monoclonal antibody. Supernatants were applied to the wells and, after washing, an HRP-conjugated specific antibody was added to the wells. Following the next wash, color development was proportional to the protein concentration and calculated by comparison with a standard. A colorimetric method was applied to quantify total protein amount in the lavage fluids. The bicinchoninic acid (BCA) assay was available in kit form from Pierce (Rockford, IL, USA) and used according to the manufacturer's instructions [4 (link)]. All data from the analyzed cytokines and proteins are reported as relative expression to the total protein content. Statistical calculations were based on these values.
Nitrotyrosine elisa kit
Manufactured by Abcam
Sourced in United Kingdom
The Nitrotyrosine ELISA Kit is a quantitative in vitro diagnostic assay designed to measure the levels of nitrotyrosine, a biomarker for oxidative stress, in biological samples such as cell lysates, tissue homogenates, and body fluids. The kit utilizes the enzyme-linked immunosorbent assay (ELISA) principle to detect and quantify nitrotyrosine concentrations.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti cleaved parp1, by Abcam (1 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Hydrogen peroxide colorimetric fluorometric assay kit, by Abcam (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Anti fibronectin, by GeneTex (1 mentions)
Peroxidase affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Lucigenin, by Merck Group (1 mentions)
Anti p38, by Abcam (1 mentions)
Anti jnk, by Abcam (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti mmp9, by Proteintech (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti mmp 3, by Abcam (1 mentions)
Anti nf kb p65 phospho s536, by Abcam (1 mentions)
Anti alpha smooth muscle actin, by Abcam (1 mentions)
Superoxide dismutase assay kit, by Cayman Chemical (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (1 mentions)
Griess reagent, by Merck Group (1 mentions)
Anti β catenin, by Abcam (1 mentions)
8 protocols using nitrotyrosine elisa kit
1
Cytokine and Protein Analysis in Synovial Fluid
2
Oxidative Stress Biomarker Assay Protocol
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Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).
3
Inflammatory and Oxidative Stress Profiles in TNFi Therapy
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The inflammatory profile was analyzed in the serum of HD and RA patients both, before and after 6 months of TNFi therapy, by using a multiplex-type immunoassay—Bioplex (Bio-Rad, CA, USA)—in which a panel of 27 cytokines was evaluated.
Oxidative stress parameters were determined through the evaluation of oxidation of both lipids and proteins, along with the analysis of the total antioxidant capacity. Assays of lipid peroxidation levels were carried out using the Thiobarbituric acid reactive substances (TBARS) assay kit (Canvax Biotech, Córdoba, Spain), following the manufacturer's recommendations.
Protein nitrosylation was measured by using the Nitrotyrosine ELISA kit (Abcam, Cambridge, UK), following the manufacturer's recommendations. Serum total antioxidant capacity (TAC) was measured by quantitative colorimetric determination, using TAC Assay kit (Biovision, Mountain View, CA, USA) following the instructions provided by the manufacturer.
Oxidative stress parameters were determined through the evaluation of oxidation of both lipids and proteins, along with the analysis of the total antioxidant capacity. Assays of lipid peroxidation levels were carried out using the Thiobarbituric acid reactive substances (TBARS) assay kit (Canvax Biotech, Córdoba, Spain), following the manufacturer's recommendations.
Protein nitrosylation was measured by using the Nitrotyrosine ELISA kit (Abcam, Cambridge, UK), following the manufacturer's recommendations. Serum total antioxidant capacity (TAC) was measured by quantitative colorimetric determination, using TAC Assay kit (Biovision, Mountain View, CA, USA) following the instructions provided by the manufacturer.
4
Quantification of Peroxynitrite-Induced Protein Damage
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The level of 3-nitrotyrosine- (3-NT-) modified proteins was used as marker of protein damage induced in HUVEC by peroxynitrite and quantified using nitrotyrosine ELISA kit (Abcam ab113848, UK).
After treatments with n-VLDL or ox-VLDL at different concentration (75, 140 μg/mL), HUVEC were trypsinized, pelleted at 500 ×g for 10 min, and washed twice using PBS buffer. Then, cells (~1 × 106) were suspended in sample extraction buffer and incubated on ice for 20 min. After centrifugation at 12,000 ×g 4°C for 20 min, the 3-NT levels were determined colorimetrically according to the manufacturer's instructions.
After treatments with n-VLDL or ox-VLDL at different concentration (75, 140 μg/mL), HUVEC were trypsinized, pelleted at 500 ×g for 10 min, and washed twice using PBS buffer. Then, cells (~1 × 106) were suspended in sample extraction buffer and incubated on ice for 20 min. After centrifugation at 12,000 ×g 4°C for 20 min, the 3-NT levels were determined colorimetrically according to the manufacturer's instructions.
5
ELISA Analysis of Plasma NE and 3-NT
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ELISA analysis was conducted according to the manufacturer’s instructions. The plasmic NE concentration in the rats was measured by using NE ELISA kit (E-EL-0047c, Elabscience). 3-NT concentration in the supernatant of culture medium of microglia was measured using Nitrotyrosine ELISA kit (ab113848, Abcam).
6
Quantifying 3-Nitrotyrosine Modified Proteins
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The level of 3-nitrotyrosine modified proteins was determined using Nitrotyrosine ELISA Kit (Abcam Cambridge, UK, ab113848) following the manufacturer’s instructions with slight modifications. The amount of 0.1 g of leaf tissue was homogenized in 150 µl of 50 mM Tris–HCl buffer, pH 7.5 with 1 mM PMSF. The extract was centrifuged on QIAshredder Mini Spin Column (Qiagen) at 14,000×g for 10 min at 4 °C and the supernatant was used for further measurements. The assay was performed as an end-point measurement at 450 nm, after termination the reaction by adding 100 ul of 1 N HCl. Results were normalized considering the total protein content in the homogenized samples and expressed as ng 3NT-BSA per µg of protein. The sample protein concentration in the extract was determined by the Bradford’s method109 (link). Spectrophotometrical measurements were performed using the Synergy HTX Multi-Mode Reader (BioTek) in 3 biological replicates.
7
Muscle Stem Cell Oxidative Stress Analysis
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Satellite cells prepared from mdx;STOPFloxBmi1 EDL muscles were transfected with A-Cre to overexpress Bmi1 and a proportion then treated with LV-shMt1. Cells infected with A-GFP were used as controls. Proteins were extracted from the cells and concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). The amount of 3-NT protein in the lysates was determined by the Nitrotyrosine ELISA kit (Abcam) according to the manufacturer’s protocol. The OD was recorded at 450 nm and the data were analyzed with the program XLfit 5.3.1.3 (IBDS).
8
Quantifying Oxidative Protein Damage in Corals
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3-nitrotyrosine modification, a well-established marker of protein damage, is a product of PTN resulting from oxidative damage to proteins by peroxynitrite which is formed in vivo by the reaction of nitric oxide and superoxide anion35 (link). The level of nitrotyrosine-modified proteins in corals was determined using the commercial kit “Nitrotyrosine ELISA Kit” (ab113848 Abcam) following manufacturer’s instructions. Briefly, the provided microplates are evenly coated with a nitrotyrosine containing antigen, and competitive ELISA is performed by adding the test sample mixed with the provided HRP conjugated anti-3NT antibody. A standard curve is generated from provided standard (3NT BSA standard) for accurate quantification of the 3NT content in the test samples. The assay was followed by monitoring the HRP-dependent color change in each well at 600 nm using a spectrofluorometer (Xenius®, SAFAS, Monaco). Data were normalized considering the total protein content in the sample homogenates in each well and expressed as ng NT-BSA mg protein−1.
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