The control and SIN-1 treated IEC-18 cell lysates were subjected to immunoprecipitation with 10 μg of anti-nitro tyrosine antibody (Abcam, Cambridge, MA, USA). Pierce classic IP kit was used to immobilize the anti-nitrotyrosine IgG to the resin and the immunoprecipitated proteins were eluted with Laemmli sample buffer. The samples obtained from immunoprecipitation were subjected to immunoblotting and probed for SGLT1. The secondary detection was performed as described in Western blot analysis and images were recorded.
Anti nitrotyrosine antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-nitrotyrosine antibody is a lab equipment product used to detect and quantify the presence of nitrotyrosine, a post-translational modification that occurs in proteins due to nitric oxide-derived oxidants. This antibody can be used in various techniques such as western blotting, immunohistochemistry, and ELISA to study the role of nitrotyrosine in biological processes.
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Lab products found in correlation
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Mtt cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
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Cisplatin, by Merck Group (1 mentions)
Cyclin b1, by Santa Cruz Biotechnology (1 mentions)
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Oxiselect advanced glycation end product age competitive elisa kit, by Cell Biolabs (1 mentions)
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Optiview dab ihc detection kit, by Roche (1 mentions)
Anti f4 80 antibody, by Thermo Fisher Scientific (1 mentions)
Substrate chromogen system, by Agilent Technologies (1 mentions)
Diaminobenzidine dab, by Agilent Technologies (1 mentions)
Anti ly6g antibody, by BD (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Cleaved il 1β, by Cell Signaling Technology (1 mentions)
Total enos, by Cell Signaling Technology (1 mentions)
8 protocols using anti nitrotyrosine antibody
1
Nitrosylated SGLT1 Protein Detection
2
Nitric Oxide Modulation in Cell Proliferation
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Polyclonal rabbit anti-human iNOS antibody (dilution1:200) was obtained from Santa Cruz Biotechnology. Anti-nitrotyrosine antibody was purchased from Abcam. The 10% acrylamide gels for western blot were purchased from Bio-Rad Laboratories. DETA NONOate (nitric oxide donor) and cisplatin were purchased from Sigma. MTT Cell Proliferation Assay Kit was purchased from Life Technologies.
3
Quantifying Superoxide and Nitrosative Stress
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Superoxide levels in heart tissue were quantified using high-performance liquid chromatography (HPLC)-based detection of dihydroethidium (DHE) oxidation products [23] (link). Briefly, hearts harvested from mice were cut into small pieces, weighed and immediately incubated with DHE (100 μM in PBS) at 37 °C for 30 min. The sample was then washed once with PBS. DHE and its oxidation products were extracted with acetonitrile (500 μl), sonicated (3 × 30 s, 8 W), centrifuged (14,000 rpm, 10 min at 4 °C) and the supernatant dried under vacuum. The samples were further dissolved in 120 μl PBS-DTPA and injected into the HPLC system. The specific superoxide oxidation product, 2-hydroxyethidine (EOH), was quantified by comparison of peak signal between the samples and standard solutions under identical chromatographic conditions. Results are expressed as the ratio of EOH generated per DHE consumed (initial DHE concentration minus remaining DHE; EOH/DHE).
As a second method, we quantified tyrosine nitrosylated proteins as an index of nitroso-oxidative modification, by immunoblotting using an anti-nitrotyrosine antibody (Abcam; 1:3000). Levels were normalized by the level of actin in the same samples.
As a second method, we quantified tyrosine nitrosylated proteins as an index of nitroso-oxidative modification, by immunoblotting using an anti-nitrotyrosine antibody (Abcam; 1:3000). Levels were normalized by the level of actin in the same samples.
4
Antibody-based Protein Analysis Protocol
5
Antioxidant and Oxidative Stress Assays
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2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), hydrogen peroxide, nicotinamide adenine dinucleotide (NADH), reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), sodium pyruvate, thiobarbituric acid (TBA), trichloroacetic acid (TCA), 2,4-dinitrophenylhydrazine (DNPH), anti-DNP antibody produced in rabbit, anti-rabbit IgG (whole molecule)–peroxidase antibody produced in goat, SOD Determination Kit, and Sigma Fast™ OPD Tablet Sets were purchased from Sigma-Aldrich Chemical Co., Warsaw, Poland. Pierce™ BCA Protein Assay Kit and rabbit anti-goat IgG secondary antibody biotin were from Thermo Fisher Scientific, Waltham, MA, USA. OxiSelect™ Advanced Glycation End Product (AGE) Competitive ELISA Kit was obtained from Cell Biolabs, San Diego, US. Anti-nitro tyrosine antibody and streptavidin (HRP) were purchased from Abcam, Cambridge, Great Britain. Other chemicals, all of the analytical grade, were obtained from POCh, Gliwice, Poland.
6
Renal Macrophage and Neutrophil Quantification
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Paraffin-embedded 5-µm kidney sections were deparaffinized and rehydrated. Endogenous peroxidase activity was first quenched by H2O2 peroxidase blocking reagent (DakoCytomation). Macrophages and neutrophils were detected by using an anti-F4/80 antibody (1:50; eBioscience) and an anti–Ly-6G antibody (1:50; BD Biosciences), respectively; for 30 minutes at room temperature (RT). Sections were then washed and incubated with a secondary biotinylated goat anti-rabbit antibody (1:500; Jackson Immunoresearch) for 30 minutes at RT. Streptavidin-HRP was added and coloration was revealed using diaminobenzidine (DAB) with the substrate chromogen system from Dakocytomation. The number of F4/80+ and Ly-6G+ cells was counted in 10 non-overlapping fields (x400 magnification). Nitrotyrosine staining was performed using an anti-nitrotyrosine antibody (1:400; Abcam) and the OptiView DAB IHC Detection Kit (Ventana Medical Systems) according to manufacturer’s instructions. Nitrotyrosine intensity in the renal cortex was quantified using NIH ImageJ software.
7
Western Blot Analysis of Protein Expression
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Protein samples with equal amount of total protein (20 μg) were separated on SDS-PAGE (8–15%). The separated protein gel was then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature for 1 h in Tris-buffered saline containing 0.1% Tween-20, primary antibody incubation (TXNIP, Cat#: 14715; Thioredoxin 1, Cat#: 2285; GLUT4, Cat#: 2213; pyruvate dehydrogenase E1-alpha [PDH E1α], Cat#: 31866; p-eNOS, Cat#: 9571; Total-eNOS, Cat#: 5880; NLRP3, Cat#: 13158; VCAM-1, Cat#: 13662; ICAM-1, Cat#: 4915; Cleaved-IL-1β, Cat#: 83186 and GAPDH, Cat#: 5174 were all from Cell Signaling Technology; Anti-Nitro tyrosine antibody [Cat#: ab42789, Abcam Company]) was carried out overnight at 4°C. Afterward, secondary antibody incubation with a peroxidase-conjugated AffiniPure goat anti-rabbit or anti-mouse IgG was conducted for 90 min at room temperature. After washing 3 times, the membranes were subjected to ECL detection. Densitometric analysis was performed using the Tanon Gel Imaging System (Shanghai Tanon, Shanghai, China). The housekeeping gene GAPDH served as a loading control.
8
Immunohistochemical Analysis of Rat IVD
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Deparaffinized sections of rat IVD were stained with hematoxylin and eosin (H&E). The sections were stained with anti-nitrotyrosine antibody (diluted 100-fold; Abcam, Cambridge, UK), followed by staining with horseradish peroxidase (HRP)-conjugated goat antimouse IgG (diluted 200-fold; Sigma-Aldrich), anti-TNFα antibody (diluted 100-fold; Novus Biologicals, Littleton, CO, USA), and anti-IL-1β (diluted 100-fold; Bioworld Technology, St. Louis Park, MN, USA), followed by HRP-conjugated goat antirabbit IgG (diluted 200-fold; Sigma-Aldrich). Staining was visualized using diamino benzidine (nacalai tesque, Kyoto, Japan). Nuclei were stained with hematoxylin. Antigen retrieval was achieved by pressure-cooking in citrate buffer (pH 6.0) for 20 minutes. All specimens were viewed under a microscope (BZ-9000; Keyence Co., Osaka, Japan). The frequency of nitrotyrosine-positive cells was measured using Image Processing and Analysis in Java at a magnification of × 200. We chose three individual areas at random and calculated the average of the frequency.
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