Sudan Black, Prussian Blue, ( ±) α-LA, Luperox® DI (tert-Butyl peroxide), anti-fatty acid synthase (FAS), and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). BODIPY® 598/591 C11, MitoTracker Deep Red FM, DAPI, were purchased from Invitrogen/Molecular Probes (Eugene, OR). MitoPeDPP® was purchased from Dojindo Molecular Technologies, Inc. (Rockville,MD) Anti-PANK2, anti-MTND1, anti-NDUFA9, anti-NFS1, anti-ISCU, anti-LYRM4anti-NRF2, PDH hand complex I activity kit and aconitase kit were purchased from Abcam (Cambridge, UK), Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-alpha-aminoadipic semialdehyde synthase (AASS), anti-FOXN4, anti-hnRNPA/B,anti-NF-Y, anti-Tau, anti-GPX4 and anti-AASDHPPT were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). Anti-PLA2G6 and anti-SOD were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-actin was acquired from MyBiosource (San Diego, California, USA). OxyBlot Protein Oxidation Detection Kit was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).
Anti sod
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-SOD is a laboratory reagent produced by Santa Cruz Biotechnology. It is a specific antibody that can be used to detect and quantify the presence of superoxide dismutase (SOD) proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Facsscan, by BD (3 mentions)
Anti ho 1, by Santa Cruz Biotechnology (2 mentions)
Anti hnrnpab, by Thermo Fisher Scientific (1 mentions)
Anti ndufa9, by Abcam (1 mentions)
Mitopedpp, by Dojindo Laboratories (1 mentions)
Anti mt nd1, by Abcam (1 mentions)
Oxyblot protein oxidation detection kit, by Merck Group (1 mentions)
Sudan black, by Merck Group (1 mentions)
Immun star hrp substrate kit, by Bio-Rad (1 mentions)
Prussian blue, by Merck Group (1 mentions)
Anti pank2, by Abcam (1 mentions)
Anti tau, by Thermo Fisher Scientific (1 mentions)
Anti gpx4, by Thermo Fisher Scientific (1 mentions)
Anti pla2g6, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by MyBioSource (1 mentions)
Trypsin, by Merck Group (1 mentions)
Anti lipoic acid, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitors, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
9 protocols using anti sod
1
Mitochondrial Dysfunction Biomarker Assay
2
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared by incubation with whole-cell lysis buffer that included 0.5% NP40 and 1% SDS supplemented with HALT protease and phosphatase inhibitors (Sigma, St Louis, MO, USA). Lysates were cleared by centrifugation and protein concentration was tested by DC assay (Bio-Rad, Shanghai, China ). Lysates were boiled with SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore, Guangzhou, China). Membranes were blocked in 5% nonfat dry milk TBS-T (10 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 0.1% Tween 20) buffer and incubated in primary antibodies diluted in blocking buffer at 4 °C overnight. Blots were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 10 000; GE Healthcare, Shanghai, China) in blocking buffer at room temperature. Immune complexes were visualized with an enhanced chemiluminescence kit (GE Healthcare). Primary antibodies for immunodetection were sourced as follows: anti-tubulin (goat, Santa Cruz Biotechnology, Guangzhou, China), anti-DDX59 (Abcam, Guangzhou, China), anti-GAPDH (Bethyl, Beijing, China), anti-Lamin B1 (Santa Cruz Biotechnology), anti-SOD (Santa Cruz Biotechnology).
3
Western Blot Analysis of Cellular Proteins
4
Evaluating Antioxidant Enzyme Expression
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To evaluate HO-1, NQO1 and SOD expression, IEC-6 cells (2 × 104 cells/well; 96-well plates) were treated with IS (31.2–250 μM) for 24 h.
Thereafter, the cells were collected, washed with PBS, and incubated with Fixing Solution for 20 min at 4 °C and successively for 30 min at 4 °C with Fix Perm Solution. Anti-HO-1 (sc-10789, Santa Cruz Biotechnology) or anti-NQO1 (sc-376023, Santa Cruz Biotechnology) or anti-SOD (sc-30080, Santa Cruz Biotechnology) antibody were subsequently added. The secondary antibody was added, for 30 min, in Fix Solution, and cell fluorescence was evaluated by FACSscan (Becton Dickinson) and elaborated with Cell Quest software, as previously reported [60 (link)].
Thereafter, the cells were collected, washed with PBS, and incubated with Fixing Solution for 20 min at 4 °C and successively for 30 min at 4 °C with Fix Perm Solution. Anti-HO-1 (sc-10789, Santa Cruz Biotechnology) or anti-NQO1 (sc-376023, Santa Cruz Biotechnology) or anti-SOD (sc-30080, Santa Cruz Biotechnology) antibody were subsequently added. The secondary antibody was added, for 30 min, in Fix Solution, and cell fluorescence was evaluated by FACSscan (Becton Dickinson) and elaborated with Cell Quest software, as previously reported [60 (link)].
5
Evaluation of Inflammatory Markers in IEC-6 Cells
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IEC-6 cells were plated into 96-well plates (2 × 103 cells/well) and treated with the peptides in inflammatory conditions, as previously described, for 24 h in order to evaluate COX-2, iNOS, HO-1, and SOD expression and nitrotyrosine formation. For this analysis, the cells were collected and washed with phosphate-buffered saline (PBS). Fixing solution was added to cells for 20 min and then incubated in fix/perm solution for a further 30 min. Anti-COX-2 (BD Transduction Laboratories, Milan, Italy), anti-iNOS (BD Transduction Laboratories, Milan, Italy), anti-HO-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-SOD (Santa Cruz Biotechnologies, Dallas, TX, USA), and anti-nitrotyrosine (Merck Millipore, Milan, Italy) antibodies were then added for 1 h. The secondary antibody, in fixing solution, was added to the cells and cell fluorescence was then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy) [35 (link)].
6
Quantification of Liver Protein Expression
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Cytoplasmic and nuclear extracts were prepared from liver homogenates[39 (link)]. The supernatant fraction was collected, aliquoted, and stored at −80 °C. Protein content was determined by Bradford’s method[32 (link)]. Lysed proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes[40 (link),41 (link)]. The membranes were then blocked with 5% skim milk in Tris buffer containing 0.05% Tween (TTBS) for 1 hour at room temperature and probed overnight at 4 °C with anti-Nrf2 (57 kDa), anti-Keap1 (69 kDa), anti-NQO1 (31 kDa), anti-SOD (32 kDa), anti-TLR4 (95 to 120 kDa), anti-NFκB (65 kDa), anti-COX-2 (21 kDa), and anti-iNOS (120 kDa) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, United Staes), diluted 1:200 to 1:1000 with TTBS in dehydrated milk at 5%. Primary antibodies were detected with HRP-conjugated anti-rat IgG, anti-rabbit IgG, or anti-goat IgG secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Protein detection was performed with a commercially available electrochemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, Bucks, England)[42 (link)]. Density of the specific bands was quantified with imaging densitometry software (Scion Image, Scion Corporation, Frederick, MA, United States).
7
Western Blot Analysis of Antioxidant Proteins
8
Ghrelin's Impact on Cellular Oxidative Stress
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Ghrelin was purchased from Sigma Chemical (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO) (Sigma, St. Louis, MO). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Gibco (Grand Island, NY, USA). Medium 199 was obtained from Sigma-Aldrich (St. Louis, MO). Annexin V-FITC and propidium iodide (PI) were obtained from Becton Dickinson (Mountain View, CA, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and H2DCFDA were obtained from Beyotime (Beyotime Institute of Biotechnology, Shanghai, China). Anti-SOD, anti-CAT, and anti-MDA antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). SOD, CAT, and MDA colorimetric kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
9
Characterization of Inflammatory Modulators
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J774A.1 cells were plated into 96-well plates (5 × 104 cells/well) and treated with 4 and 5 (200–25 µM) for 1 h and then co-exposed to LPS (1 µg/mL) for 24 h. Cells were then collected, washed twice with PBS, and then incubated in fixing solution for 20 min at 4 °C and then incubated in Fix PermSolution for 30 min at 4 °C. Anti-iNOS (e-Bioscience, Dunwoody Park, Atlanta, GA, USA), Anti-COX-2 (e-Bioscience), HO-1 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-SOD (Santa Cruz Biotechnology) antibodies were then added for a further 30 min. The secondary antibody was added in Fix Solution, and cells’ fluorescence was evaluated using a fluorescence-activated cell sorter (FAC Sscan; Becton Dickinson) and elaborated with Cell Quest software as previously described [44 ].
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