The Western blot was performed as previously described [25 (link)]. Liver protein extracts were obtained by lyses homogenized tissue in Ripa buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein samples (40 μg) were resolved on 10% or 12% Tris-HCl polyacrylamide gels and subsequently transferred to a nitrocellulose membrane. Membranes were probed with commercially available rabbit polyclonal anti-ApoE (1:500 dilution), anti-GRP-78 (1:500) (Abcam, Cambridge, MA, USA), anti-β-tubulin (1:200) (Cell Signaling, Danvers, MA, USA), anti- ERP29 and anti-α-tubulin (1:2000), anti-SREBP -1c and anti-SOD2 (1:500) antibodies (Abcam, Cambridge, MA, USA), followed by HRP-conjugated anti-rabbit antibody (1:10000) and ECL Plus detection reagents (GE Biosciences, Piscataway, NJ, USA). Relative ApoE, GRP-78, β-tubulin, α-tubulin, ERP29, SREBP and SOD2 band densities were determined by densitometrical analysis using the Image Studio Lite software from LI-COR Corporate Offices-US (Lincoln, Nebraska USA). In all instances, density values of bands were corrected by subtraction of the background values. The results were expressed as the ratio of ApoE, GRP-78, ERP29 and SOD2 to that of β-tubulin and SREBP to that of α-tubulin our β-tubulin.
Anti sod2
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-SOD2 is a primary antibody that specifically binds to and detects Superoxide Dismutase 2 (SOD2), a key antioxidant enzyme involved in the mitochondrial defense system against oxidative stress. This antibody can be used for various applications such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of SOD2 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti sod1, by Abcam (7 mentions)
Anti nrf2, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Anti β tubulin, by Cell Signaling Technology (2 mentions)
Anti ho 1, by Abcam (2 mentions)
Anti actin, by Abcam (2 mentions)
Anti catalase, by Abcam (2 mentions)
Anti gpx1, by Abcam (2 mentions)
Anti histone h3, by Cell Signaling Technology (2 mentions)
Anti sirt3, by Santa Cruz Biotechnology (2 mentions)
Anti β tubulin, by Bioworld Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Anti cat, by Abcam (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Anti apoe, by Abcam (1 mentions)
Anti srebp 1c, by Abcam (1 mentions)
Ecl plus detection reagent, by GE Healthcare (1 mentions)
30 protocols using anti sod2
1
Western Blot Analysis of Liver Proteins
2
Oxidative Stress Protein Expression
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Anti caspase 3, anti HO1, anti Nrf2, anti SOD2, anti actin, anti lamin B1 and anti GCLC antibodies were purchased from Abcam (Cambridge, MA, USA). K2Cr2O7 and all other reagents were bought from Sisco Research Laboratory (Mumbai, India).
3
Protein Extraction and Western Blot
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Protein extraction was carried out by lysing cells in PBS buffer containing 1% Triton X-100 with added protease and phosphatase inhibitors (Roche). Protein content was determined using Bradford assay (Pierce). Approximately 25 to 70 μg of total protein was resolved on 8, 10 or 12% SDS-PAGE gel before transferring onto PVDF membrane (Millipore) for detection using ECL reagent (Pierce). The following antibodies were used: anti-phospho-JNK, anti-JNK, anti-phospho-MKK4, anti-MKK4, anti-FTH1 (Cell Signaling Technology), anti-TRX (Santa Cruz), anti-LC3B (Abgent), anti-SOD2 (Abcam), anti-actin and HRP-conjugated anti-rabbit and anti-mouse (Sigma).
4
SOD2 Acetylation Quantification in Kidney Tissue
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Kidney tissue lysate (0.5 mg) was pre-cleaned with 20 μl of A/G plus agarose (Cat. No. sc-2003; Santa Cruz Biotechnology) and precipitated by 20 μl of anti-SOD2 (Abcam Cat# ab13533, RRID:AB_300434 ) at 4°C overnight. Following further precipitation with 20 μl of A/G plus agarose for 2 h, samples were washed three times using cold PBS, solubilized with 40 μl 2 × SDS sample buffer (Cat. No. S3401–1VL; Sigma-Aldrich). An equal amount of eluted protein was resolved on the gel for assessing the acetylation status SOD2 using anti-Ac-lysine (AKL5C1) antibody (Santa Cruz Biotechnology, Cat# sc-32268, RRID:AB_627898 ).
5
Western Blotting of SOD2 and NDUFV2
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Western blotting was performed as described previously [17 (link)], with modifications. Commercial polyclonal anti-SOD2 (Abcam, Cambridge, MA, USA) and polyclonal anti-NUDFV2 (Abcam) were used, and monoclonal anti-α-tubulin (Abcam) was used as a control. Briefly, the samples were washed by centrifugation in DPBS at 10,000 × g for 5 min. The pellets were re-suspended with lysis buffer containing 5% 2-mercaptoethanol and incubated for 10 min at RT. The samples were electrophoresed on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Amersham). The membranes were blocked with PBS-Tween containing 5% skim milk powder (blocking solution) for 3 h at RT. After washing, the membranes were incubated overnight with anti-NDUFV2 (1:3,000) and anti-SOD2 (1:5,000) diluted with blocking solution. Then, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit immunoglobulin G (1:3,000, Abcam) for 1 h. After the membranes were washed, proteins were detected by enhanced chemiluminescence reagents. All bands were scanned with a GS-800 Calibrated Imaging Densitometer (Bio-Rad) and analyzed with Quantity One (v.4.6, Bio-Rad). The signal intensity ratios of the bands were calculated for SOD and NUDFV2/α-tubulin.
6
Quantifying SOD2 mRNA and Protein
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Total RNA from cells was extracted by Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The mRNA was measured by quantitative real-time PCR (ABI 7500) after reverse transcription, and we chose β-actin as an internal quantitative control for samples. The β-actin as the internal reference, and relative quantification of SOD2 mRNA was calculated by the 2-ΔΔCt method [(SOD2 Ct- β-actin Ct in rs4880 C allele) − (SOD2 Ct- β-actin Ct in rs4880 T allele)].
For Western blot analyses, cells were lysed with Radio Immunoprecipitation Assay (RIPA) lysis buffer. Twenty-five milligrams of total protein were electrophoresed on a 12% SDS-PAGE gel, transferred to a poly vinylidene fluoride (PVDF) membrane (GE Healthcare Life Sciences, Marlborough, MA, USA), and blocked with 10% non-fat milk in Tris-buffered saline and Tween-20 (TBST). The PVDF membrane was incubated with anti-SOD2 (1:1000; Abcam, Cambridge, MA, USA) and anti-β-actin antibody (1:1000; Abcam) overnight at 4 °C to confirm equivalent protein loading in each lane, next with Horseradish Peroxidase (HRP)-conjugated rabbit-anti-mouse IgG for 1 h at room temperature, washed 3 times with TBST for 15 min each, and finally developed with enhanced chemiluminescence (ECL) (Amersham Life Science, Madison, WI, USA) on X-ray film.
For Western blot analyses, cells were lysed with Radio Immunoprecipitation Assay (RIPA) lysis buffer. Twenty-five milligrams of total protein were electrophoresed on a 12% SDS-PAGE gel, transferred to a poly vinylidene fluoride (PVDF) membrane (GE Healthcare Life Sciences, Marlborough, MA, USA), and blocked with 10% non-fat milk in Tris-buffered saline and Tween-20 (TBST). The PVDF membrane was incubated with anti-SOD2 (1:1000; Abcam, Cambridge, MA, USA) and anti-β-actin antibody (1:1000; Abcam) overnight at 4 °C to confirm equivalent protein loading in each lane, next with Horseradish Peroxidase (HRP)-conjugated rabbit-anti-mouse IgG for 1 h at room temperature, washed 3 times with TBST for 15 min each, and finally developed with enhanced chemiluminescence (ECL) (Amersham Life Science, Madison, WI, USA) on X-ray film.
7
Osteogenic Differentiation and ROS Regulation
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Annexin V-FITC/PI detection kit was purchased from BD Biosciences (San Jose, CA, USA). Fluorometric Intracellular ROS Kit and N-acetyl-L-cysteine (NAC) were from Sigma-Aldrich (St. Louis, USA). Information of primary antibodies is listed as follows: anti-Runx2 (1 : 500, Abcam), anti-Osterix (1 : 500, Abcam), anti-OPN (1 : 1000, Abcam), anti-BSP (1 : 1000, CST), anti-OCN (1 : 500, Abcam), anti-NADPH oxidase 4 (NOX4) (1 : 500, Abcam), anti-SOD2 (1 : 500, Abcam), anti-Caspase-3 (1 : 1000, CST), and anti-GAPDH (1 : 5000, Bioworld Technology Inc., USA). ALP staining and alizarin red were both from Cyagen (Guangzhou, China). For semiquantitative analysis, p-nitrophenyl phosphate (p-NPP) and 10% cetylpyridinium chloride were from Sigma-Aldrich (St. Louis, USA). Alexa Fluor 488 conjugated was from Jackson ImmunoResearch (USA). DAPI was from Sigma-Aldrich (St. Louis, USA). For scaffold fabrication, gelatin, carboxymethyl chitosan, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were all purchased from Aladdin (Shanghai, China). Calcein AM (4 mM) and Lipofectamine 3000 were from Thermo Fisher Scientific (USA).
8
Liver Protein Extraction and Western Blot Analysis
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Total proteins were extracted from liver tissue using a protein extraction kit (Minute Protein Extraction Kit; Invent Biotechnologies, Inc., Eden Prairie, MN, USA). Extracted proteins were quantitated by the BCA method (BCA protein Assay Kit; Takara Bio, Inc., Otsu, Japan), and were diluted to the same protein concentrations. Equivalent amounts (50 µg) of each group were run on SDS polyacrylamide gels. Proteins were transferred electrically to a PVDF membrane and incubated with the following antibodies; anti-TNF-α, anti-IL1β (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-DMT1, anti-hepcidine25, anti-CPT1A, anti-CPT2, anti-SOD2, anti-catalase (Abcam, Tokyo, Japan), anti-GPx4 (LifeSpan BioSciences, Seattle, WA, USA). Membranes were washed and incubated with respective secondary antibodies. Detection of bands was performed by chemiluminescent analysis. Beta actin was used as a loading control. Representative data of three experiments were shown on figures.
9
Transcriptional and Translational Regulation in HAEC
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RNA isolation, reverse transcription and SYBR® green-based (Applied Biosystems) quantitative PCR was carried according to standard protocols using a Quant Studio 7 Flex Real Time PCR thermocycler (Applied Biosystems) with the associated sequence detection system and software. Expression was calculated using the ΔΔCT method. Relative gene expression was normalized to β-actin (house-keeping gene). Western blot analyses of HAEC lysates were conducted according to standard protocols using the following specific antibodies:
anti-Sirt3 (rabbit monoclonal, Cell Signalling Technology), anti-C/EBP-β [C19] (rabbit polyclonal, Santa Cruz Biotechnology), anti-SOD2 (rabbit polyclonal, Abcam), anti-catalase (mouse monoclonal, Sigma), anti-glutathion peroxidase 1 (rabbit polyclonal, Novus Biologicals), anti-total eNOS (mouse polyclonal, BD Transduction Laboratories), anti-eNOS (pThr495) (mouse, monoclonal, BD Transduction Laboratories), anti-eNOS (pSer1177) (mouse, monoclonal, BD Transduction laboratories), anti-xanthine oxidase (rabbit polyclonal, Biorbit), anti-NADPH oxidase subunit p22phox (rabbit polyclonal, Biorbit), and anti-β actin (mouse monoclonal, Sigma-Aldrich). Specific signals were detected using species-specific secondary antibodies.
anti-Sirt3 (rabbit monoclonal, Cell Signalling Technology), anti-C/EBP-β [C19] (rabbit polyclonal, Santa Cruz Biotechnology), anti-SOD2 (rabbit polyclonal, Abcam), anti-catalase (mouse monoclonal, Sigma), anti-glutathion peroxidase 1 (rabbit polyclonal, Novus Biologicals), anti-total eNOS (mouse polyclonal, BD Transduction Laboratories), anti-eNOS (pThr495) (mouse, monoclonal, BD Transduction Laboratories), anti-eNOS (pSer1177) (mouse, monoclonal, BD Transduction laboratories), anti-xanthine oxidase (rabbit polyclonal, Biorbit), anti-NADPH oxidase subunit p22phox (rabbit polyclonal, Biorbit), and anti-β actin (mouse monoclonal, Sigma-Aldrich). Specific signals were detected using species-specific secondary antibodies.
10
Oxidative Stress Protein Profiling
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Collected cells were homogenized in lysis buffer (5% SDS, 10 mM EDTA, 50 mM NaCl, 10 mM Tris–HCl). Protein concentrations were determined using pierce BCA protein assay (Thermo Fisher, Rockford, IL, USA). Proteins were resolved using SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Membranes were blocked in 5% milk, incubated with primary antibody at a concentration of 1:1000, then incubated with secondary antibody at a concentration of 1:10,000 and read using ECL regent (Bio-Rad, Richmond, CA, USA). Antibodies anti-GCLM, anti-GSS, anti-GCLC, anti-GSR, anti-GPX1, anti-SOD1, anti-SOD2, anti-TKT, anti-G6PD, anti-CGL and anti-Actin were purchased from Abcam. Anti-NRF2 and anti-CBS antibody was purchased from Cell Signaling Technology.
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