Cells were cultured in a 6-well plate. After treatment, the total protein of cells was obtained and quantified using a BCA assay (Sigma-Aldrich, St Louis, MO, USA). Proteins were separated according to their molecular weights through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). After successive incubation with primary and rabbit secondary antibody conjugated with HRP (Abcam, Cambridge, UK), membranes were observed using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Primary antibodies used were anti-GPX-1, anti-CAT, anti-SOD-1, anti-SOD-2, anti-iNOS, anti-COX-2, and anti-β-actin (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA).
Anti sod2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SOD2 is a primary antibody that specifically recognizes the SOD2 (Superoxide Dismutase 2) protein. SOD2 is an enzyme involved in the conversion of superoxide radicals into hydrogen peroxide and oxygen, playing a crucial role in cellular antioxidant defense mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Anti sod1, by Cell Signaling Technology (7 mentions)
Anti β actin, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti catalase, by Cell Signaling Technology (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Anti bcl 2, by Cell Signaling Technology (3 mentions)
Anti gpx1, by Cell Signaling Technology (2 mentions)
Anti cat, by Cell Signaling Technology (2 mentions)
Anti sirt3, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti survivin, by Cell Signaling Technology (2 mentions)
Anti atf4, by Cell Signaling Technology (2 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Anti grp94, by Cell Signaling Technology (2 mentions)
N acetyl l cysteine nac, by Merck Group (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti atf6, by Cell Signaling Technology (2 mentions)
Anti ire1α, by Cell Signaling Technology (2 mentions)
Anti sod3, by Santa Cruz Biotechnology (2 mentions)
22 protocols using anti sod2
1
Antioxidant Protein Expression Analysis
2
Acetylation Profiling in Heart Disease
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Primary antibodies included anti-acetyl-lysine (Cell Signaling #9441), anti-SIRT3 (Cell Signaling #5490), anti-SOD2 (acetyl K68) (Abcam ab137037), anti-SOD2 (Cell Signaling #13141), anti-VDAC (Cell Signaling #4866), anti-GAPDH (Cell Signaling #5174), anti-FXN (generous gift of Grazia Isaya, Mayo Clinic, Rochester MN), anti-LCAD (Abcam ab196655) and to the electron transport chain complexes: anti-CI-NDUFA9 (Abcam ab14713), anti-CII-SDHB (Abcam ab14714) and anti-CIII-UQCRFS1 (Abcam ab14746). Band intensities were measured using ImageJ (IJ1.46). To determine correlation between acetylation and heart function, relative density of acetylation for n = 3 western blot images was normalized to day 30 controls and averages for each group were used for correlation calculation.
3
Antioxidant Protein Expression Analysis
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Whole cell lysate was prepared with lysis buffer (50 mM Tris pH = 7.5, 0.2 M NaCl, 1% Tween-20, 1% NP40, 1 mM sodium orthovanadate, 2 mM β-glycerophosphate and prote-ase inhibitors). Twenty μg of protein were seperated by 10% SDS polyacrylamide gel electrophoresis (PAGE) and transferred to pol-yvinylidene difluoride membranes (Merck, Darmstadt, Germany). Membranes were blocked by 5% BSA in Tris-buffered saline (20 mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Tween-20 (TBST) and incubated with primary antibodies overnight at 4 °C. Membranes were then reacted with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, Dallas, TX, USA) and imaged by X-ray films. Primary antibodies for western blot were as follows: anti-ANT1 (NBP83928, Novus, St Charles, MO, USA), anti-ANT2 (14671, Cell Signaling, Danvers, MA, USA), anti-GPX1 (3286S, Cell Signaling), anti-SOD1 (2770S, Cell Signaling), an-ti-SOD2 (13141S, Cell Signaling), anti-Actin (sc-8432, Santa Cruz), and anti-Ku70 (SC-17789, Santa Cruz).
4
Antibody-Based Protein Extraction from Patient Tissues
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Experiments were performed as previously described [14 (link)]. If not declared otherwise, 3 days before harvesting, cells were transfected with siRNA as indicated. The following antibodies were used: anti-KMT9α (#27630, lot 20062017, Schüle Lab), anti-KMT9β (#28358, lot 27022018, Schüle Lab), anti-H4K12me1 (#27429, lot 27062017, Schüle Lab); anti-H4 (ab31830, lot GR3204774-2, abcam); anti-Tubulin (alpha tubulin, #T6074, lot 03714804 V, Sigma), anti-LMNA (sc-20680, lot F2607, Santa Cruz); anti-GAPDH (MAB574, lot 3273148, R&D systems); anti-SMARCA2 (NB100-55308; lot A1; Novus biologicals); anti-TIMP2 (CST#5738, lot 3, Cell signaling); anti-SOD2 (CST#13194, lot 1, Cell signaling); anti-YES1 (#PA5-80243, lot VA2919193, Invitrogen). Proteins from patient tissues were extracted using the Minilys homogenizer (Bertin instruments) and RIPA buffer (1 mM EDTA, 50 mM Tris–HCL pH7.5, SDS 0.1%, NaCl 150 mM, NP-40 1%, Na deoxycholate 1%, protease inhibitor cocktail EDTA-free). Samples were cycled for 15 s at top speed.
5
Western Blot Analysis of Antioxidant Enzymes
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Protein extracts were obtained by adding sample buffer to the cells, which were scraped and lysed by pipetting and boiling. Equal amounts of protein were loaded and separated in a SDS-PAGE; then, proteins were transferred onto a nitrocellulose membrane and incubated with the proper primary antibody. Membranes were blocked using 7.5% milk in TBS/T buffer. Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam). Membranes were washed three times with PBS/T and incubated with HRP coupled secondary anti-mouse or rabbit antibodies (Santa Cruz Biotechnologies). Membranes were finally developed using the ChemoLuminiscent Reagent (Merck Millipore, Billerica, MA, USA) accordingly to the manufacturer's instructions. Densitometric analysis was performed using the ImageJ program ver.1.48h3, National Institutes of Health (NIH).
6
Analyzing Antiviral Protein Expression
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Cultured epithelial cells after stimulation were rinsed with DPBS and proteins were extracted using radio-immunoprecipitation assay (RIPA) buffer (GenDEPOT). Denatured proteins (25 μg) were fractionated on SDS-PAGE and the gels were transferred onto a polyvinylidene difluoride membrane (Bio-Rad, MA, USA). Thereafter the membranes were rinsed and probed with the primary antibodies in the refrigerator overnight at 4 °C; the primary antibodies employed for Western blots were anti-β actin (1:5000) which was obtained from Santa Cruz, USA, anti-viperin, anti-OAS, anti-Mx, anti-TLR3, anti-RIG1, anti-MDA5, anti-SOD1, anti-SOD2, anti-IRF3, and anti-phospho-IRF3 which were obtained from Cell Signaling Technology and used at a dilution of 1:1000.
7
Antioxidant Effects of Korean Red Ginseng
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Korean Red Ginseng powder was kindly provided by the Korea Ginseng Corporation (Daejeon, Korea). The KRG powders were dissolved in distilled water. N-acetyl-l -cysteine (NAC) and Anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase-3, anti-caspase-9, anti-cleaved PARP, anti-BIM, anti-Noxa, anti-survivin, anti-IRE1α, anti-phospho IRE1α, anti-GRP94, anti-eIF2α, anti-phospho eIF2α, anti-ATF6, anti-PERK, anti-phospho PERK, anti-Bip anti-XBP1s, anti-ATF4, anti-SOD2, anti-SOD1, anti-catalase, anti-NOX4, and anti-NOX2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Bak, anti-BAX, anti-Bcl-2, anti-SOD3, and anti-CHOP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-mouse and Rabbit IgG HRP, the secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Doxorubicin-Induced Oxidative Stress Mitigation
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Doxorubicin (#5927, cell signaling), hydroxytyrosol (H4291 Sigma-Aldrich, Missouri, MO, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) (Sigma-Aldrich). Antibodies: anti-cleaved caspase-3 (#9662), anti-SOD2 (#13194), anti-Bax (#2774), anti-Bcl-2 (#15071), anti-phospho-p38 MAPK (#9211), anti-p-38 (#9212), anti-phospho-histone H2AX (Ser139) (#80312), Anti-GAPDH (#97166), anti-tubulin (#2146) (Cell Signalling Technology, Danvers, MA, USA). Secondary antibodies: anti-mouse IgG (#7076), anti-rabbit IgG (#7074) (Cell Signalling Technology).
9
Western Blot Analysis of Mitochondrial Proteins
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HRECs were lysed in cell lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1-mM phenylmethylsulfonyl fluoride and a protease/phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA). Total protein concentrations were measured using a bicinchoninic acid assay (Beyotime Biotechnology). Protein samples (30–50 µg) were used for western blot using 4% to 20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a 0.22-µm polyvinylidene fluoride membrane (Roche). After being blocked in nonfat milk, the membranes were incubated in the primary antibody at 4°C overnight and in the secondary antibody (The Jackson Laboratory, West Grove, PA, USA) at room temperature for 1 hour. The ECL Plus HRP substrate (EMD Millipore, Burlington, MA, USA) and Amersham Imager 600 (General Electric, Boston, MA, USA) were used to visualize the immunoreactive bands. The primary antibodies were as follows: anti-UCP2 (#89326), anti-SIRT3 (#2627), anti-SOD2 (#13141), anti-P21 (#2947), and anti-Actin (#5125), all of which were purchased from Cell Signaling Technology (Danver, MA, USA), and Anti-beta Tubulin antibody (#ab6046), which was purchased from Abcam (Cambridge, UK).
10
Western Blot Analysis of Oxidative Stress Markers
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A total of 2 × 106 cells were seeded in 100 mm dish. Cells were treated with RIPA buffer (Cell Signaling) for lysis. Proteins (100 μg) were separated through SDS-polyacrylamide gel, followed by transfer to polyvinylidene difluoride (PVDF) membrane (Millipore Corp). PVDF membranes were coated with anti-HO-1 (Cat# 5061), anti-Nrf2 (Cat# 12721), anti-Keap1 (Cat# 4678), anti-Bcl-xL (Cat# 2764), anti-Bcl-2 (Cat# 2876), anti-GAPDH (Cat# 5174), anti-cleaved Caspase-3 (Cat# 9661), anti-γH2AX (Cat# 2577), anti-SOD-2 (Cat# 13141), anti-Sp-1 (Cat# 5931), or anti-β-actin (Cat# 3700) (all from Cell Signaling); and then incubated in the presence of peroxidase-linked (HRP) secondary antibody. Protein bands were visualized through chemiluminescent substrate (Pierce)5 (link),15 (link).
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