Following plasma treatment, E. coli bacteria were centrifuged, suspended in PBS and sonicated three times for 5 seconds. After 10 min of centrifugation at 14,000 g, the supernatant was recovered. Total protein concentration was evaluated using the DC protein assay kit (Biorad, Marnes la Coquette, France). Carbonylated proteins were detected and analyzed after the derivatization of protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) (Protein Oxidation Detection Kit, OxyBlot™, Millipore, Molsheim, France). Derivatized samples were resolved by SDS-PAGE in a 4–20% acrylamide gel and electrotransferred onto a Hybond nitrocellulose membrane (GE Healthcare, Piscataway, NJ, USA). The primary antibody for Western blotting was raised against dinitrophenylhydrazone, and primary antibody binding was detected with a peroxidase-conjugated secondary antibody and a chemiluminescent substrate (ECL plus Western blotting detection system, GE Healthcare). Western blots were quantified using Image J. Anti-3nitrotyrosine was used to detect nitrated proteins (Abcam, Paris, France).
Anti 3 nitrotyrosine
Manufactured by Abcam
Sourced in France, United States
Anti-3-nitrotyrosine is a primary antibody that targets 3-nitrotyrosine, a post-translational modification that occurs in proteins as a result of nitrative stress. This antibody can be used to detect and quantify the presence of 3-nitrotyrosine in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl plus western blotting detection system, by GE Healthcare (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Anti matrix metalloproteinase 9 mmp 9, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Nlrp3, by Abcam (1 mentions)
Permethrin, by Merck Group (1 mentions)
Anti hmgb1, by Santa Cruz Biotechnology (1 mentions)
Pierce lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Anti bdnf, by Cell Signaling Technology (1 mentions)
Prolong diamond antifade mounting media with dapi, by Thermo Fisher Scientific (1 mentions)
Anti il 18, by Abcam (1 mentions)
Streptavidin hrp vectastain elite abc kit, by Vector Laboratories (1 mentions)
Fluorescence conjugated alexa fluor secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti tmem119, by Abcam (1 mentions)
4 protocols using anti 3 nitrotyrosine
1
Oxidative Stress Analysis in E. coli
2
Protein Extraction and Western Blot Analysis
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The extraction of tissue protein in ipsilateral hemispheres tissues was performed following the instructions of the reagents kit and using RIPA buffer. The concentrations of protein were detected by the BCA Protein Assay Kit (Sigma-Aldrich). After divorced by the SDS-PAGE, the protein samples were transferred to a PVDF filter membrane. Following being blocked with 5% nonfat milk, primary antibodies (anti-phosphorylated nNOS at Ser1214, Sigma-Aldrich; anti-3-nitrotyrosine, Abcam; anti-matrix metalloproteinase-9 (MMP-9), Abcam; ZO-1, Invitrogen, USA; NLRP3, Abcam; IL-1β, Abcam) were incubated respectively and followed by incubation with species-specific secondary antibodies (goat anti-mouse IgG 1:2000, Santa Cruz Biotechnology; goat anti-rabbit IgG 1:2000, Santa Cruz Biotechnology). Densitometry analysis was performed with Image J software after normalization to GAPDH.
3
Neuro-Inflammatory Markers and Microbiome Analysis
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We purchased PB and permethrin from Sigma-Aldrich (St. Louis, MO). Anti-RAGE, anti-Claudin 5, anti-HMGB1, anti-IL-1β, and anti-ASC-2 were purchased from Santacruz Biotechnology (Dallas, TX), Anti-BDNF from cell signaling technology (Danvers, MA), while anti-NLRP3, anti-3-nitrotyrosine, anti-IL-6, anti-IL-18, anti-TMEM 119 primary antibodies were purchased from Abcam (Cambridge, MA). Species specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA). Fluorescence conjugated (Alexa Fluor) secondary antibodies, ProLong Diamond antifade mounting media with DAPI and Pierce LAL chromogenic endotoxin quantitation kit were bought from Thermo Fisher Scientific (Waltham, MA) while enzyme-linked immunosorbent assay (ELISA) kits were purchased from ProteinTech (Rosemond, IL). Unless otherwise specified, all other chemicals used were purchased from Sigma. Paraffin-embedding of tissue and sectioning were done by AML laboratories (Baltimore, MD) and at the Instrument Resources Facility, University of South Carolina School of medicine (Columbia, SC). Microbiome analysis was done by Cosmos ID (Rockville, MD).
4
Quantification of Oxidative Stress Markers
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Whole cell or tissue samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX) with protease inhibitor cocktail (Thermo Scientific) and 40 μg total protein was used for immunoblot analysis according to our previous protocols [27] . Blots were probed with anti-Klotho (1:200; Thermo Scientific), anti-3-nitrotyrosine (1:500; Abcam, Cambridge), anti-SOD2 (1:500; Santa Cruz Biotechnology), anti-RIP1 (1:100; Santa Cruz Biotechnology), anti-RIP3 (1:500; Santa Cruz Biotechnology), anti-IL-1 beta (1:1000; Abcam), anti-tubulin (1:1000; Abcam), anti-beta-actin (1:2000; Abcam), anti-catalase (1:1000; Abcam), anti-GPX4 (1:1000; Abcam), anti-t-FoxO1 (1:1000; Cell Signaling Technology, Beverly, CA), antip-FoxO1 (1:1000; Cell Signaling Technology), anti-t-FoxO3a (1:1000; Cell Signaling Technology), anti-p-FoxO3a (1:1000; Cell Signaling Technology), or anti-GAPDH (1:1000; Cell Signaling Technology) antibodies overnight at 4 °C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signaling Technology). After washing, specific signals were determined using an ECL kit (Thermo Scientific) on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai). Gray values were analyzed with ImageJ software.
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