The method has been described previously [18 (link)]. Protein lysate from pancreatic tissue or isolated pancreatic acinar cells was prepared by homogenizing in RIPA containing protease and phosphatase inhibitor. Lysate samples (20 μg protein) were loaded on 15% polyacrylamide gel, then the gel was transferred to polyvinylidene fluoride (PVDF) membrane, and finally, blocked and incubated with primary antibody and secondary antibody. The images were detected by enhanced chemiluminescence detection system (California, USA). Anti-CBS (1 : 300) and anti-Nrf2 (1 : 1000) were purchased from Bioss (Beijing, China); anti-SIRT1 (1 : 1000) and anti-PGC1-α (1 : 1000) were purchased from Wanleibio (Shenyang, China); anti-phospho-P38 (1 : 1000), anti-P38 (1 : 1000), anti-LC3 (1 : 1000), anti-β-actin (1 : 1000), and anti-P62 (1 : 1000) were purchased from Cell Signaling Technology (Danvers, USA); anti-Parkin (1 : 1000) was from Santa Cruz Biotechnology (California, USA).
Anti nrf2
Manufactured by Bioss Antibodies
Sourced in China
Anti-Nrf2 is a laboratory reagent used for the detection and quantification of the Nrf2 protein in biological samples. Nrf2 is a transcription factor that plays a crucial role in the regulation of antioxidant and detoxification genes. Anti-Nrf2 can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and function of Nrf2 in cells and tissues.
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Lab products found in correlation
Anti bcl 2, by Bioss Antibodies (2 mentions)
Anti bax, by Immunoway (2 mentions)
Anti actin, by Abcam (2 mentions)
Anti caspase 3, by Abcam (2 mentions)
Anti ho 1, by Bioss Antibodies (2 mentions)
Anti cyp2e1, by Boster Bio (2 mentions)
Anti gapdh, by Boster Bio (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Anti lamin b, by Bioss Antibodies (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti parkin, by Santa Cruz Biotechnology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti p38, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Mitochondrial marker antibody sampler kit, by Cell Signaling Technology (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Anti hla g 4h84, by Novus Biologicals (1 mentions)
8 protocols using anti nrf2
1
Comprehensive Protein Expression Analysis
2
Western Blot Analysis of Intestinal Proteins
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Western blotting was performed using standard protocols. In brief, total proteins were extracted from small intestine tissue and mixed with loading buffer. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyacrylamide difluoride (PVDF) membranes. After blocking with 5% skim milk for 2 h at room temperature, the membranes were incubated with anti-IL-17D (dilution 1:1,000, cat. no. bs-2612R, Bioss), anti-Nrf2 (dilution 1:1,000, cat. no. ab31163, Abcam), and anti-keap1 (dilution 1:1,000, cat. no. ab139729, Abcam), or anti-β-actin (dilution 1:2,000, cat. no. bs-0061R, Bioss) and left shaking gently overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG anti-body (dilution 1:5,000; cat. no. SA00001-2, Proteintech) for 2 h at room temperature, washed in 10 mM Tris/HCl, 150 mM NaCl, and 0.05% Tween-20, pH 7.5 TBST buffer three times, and developed using enhanced chemiluminescence reagents (NCM Biotech). Densitometry values were computed using ImageJ 6.0 (National Institutes of Health) and standardized to β-actin.
3
Quantitative Gene Expression Analysis
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To quantify specific gene expression, samples were lysed in lysis buffer (Sigma-Aldrich). Samples containing equal quantities of protein were run on 10% to 12% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes, which were then blocked in 5% BSA and incubated overnight at 4°C with anti-catalase and anti-human PRL-1 (1:1,000, both from Abcam, Cambridge, UK); anti-GPX3, anti-HLAG (4h84), and anti-PGC1A (1:1,000, all from NovusBio, Littleton, CO, USA); anti-POU5F1 (1:1,000, AbFrontier, Seoul, Republic of Korea); antibodies from a Mitochondrial Marker Antibody Sampler Kit (1:1,000, Cell Signaling Technology, Danvers, MA, US); anti-HO-1 (1:500, Cell Signaling Technology); anti-ATP5B (1:500, Santa Cruz Biotechnology, Dallas, TX, USA); anti-NRF2 (1:1,000, Bioss, Woburn, MA, USA); anti-TFAM (1:1,000, Thermo Fisher Scientific, Waltham, MA, USA); and anti-GAPDH (1:3,000, AbFrontier). The membranes were then incubated with peroxidase-conjugated secondary anti-rabbit IgG and anti-mouse IgG (1:8,000, Bio-Rad). Bands were detected using enhanced chemiluminescence reagent (Bio-Rad Laboratories).
4
Western Blot Analysis of Antioxidant Proteins
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Cells were lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate). Protein lysate (30 µg) was separated on a 10% SDS-PAGE gel and then transferred to a polyvinylidene difluoride membrane (Millipore, USA) [23 (link)]. The primary antibodies used were anti-Nrf2 (1:1000; Bioss, China), anti-GR (1:1000; Boster, China), anti-catalase (1:1000; Boster, China), and anti Keap1 (1:1000; Boster, China). Anti-GAPDH antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA) was used as a loading control. After washing, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000, GE Healthcare, Little Chalfont, UK) for 1 h at room temperature. Protein bands were visualized with ECL (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) on an X-ray film, which was quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
5
Evaluating Antioxidant Protein Expressions in Chicken Kidneys
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Expressions of Nrf2, HO-1, CAT, MnSOD, Capase3, Bax, and Bcl-2 proteins in the kidneys of chickens were determined by western blot. The Protein Extraction Kit (Beyotime, Wuhan, China) was used to extract total proteins from the tissues. The BCA Protein Assay Kit (Solarbio, Beijing, China) was used to measure total protein content. The following antibodies were used to detect their respective proteins: anti-Caspase3 (1 : 1000, Abcam, Tokyo, Japan), anti-Bax (Immunoway, Suzhou, China), anti-HO-1, anti-Bcl-2, anti-Nrf2 (1 : 1000, Bioss, Beijing, China), anti-MnSOD (1 : 6000, Enzo Clinical Labs, Farmingdale, NY, USA), anti-CAT (1 : 700, Biorbyt, Cambridge, UK), anti-Actin (1 : 10000, Abcam, Tokyo, Japan). An HRP-labeled goat anti-rabbit IgG (1 : 10000, Jackson Immuno Research Labs, West Grove, PA, USA) was used as the secondary antibody. Relative band intensities were detected on a DNR Bio Imaging system by using the NcmECL Ultra method (Ncmbio, Suzhou, China). Relative intensities of these bands were normalized according to the β-actin.
6
Quantifying Nrf2 and CYP2E1 Protein Levels
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For nuclear factor erythroid 2-related factor 2 (Nrf2) expression analysis, the extraction and isolation of cytoplasmic and nuclear protein were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. For CYP2E1 expression analysis, the extraction and isolation of microsomal protein were carried out as described previously [10 (link)]. The concentration of protein was determined by BCA assay kit (Beyotime, Nanjing, China). Equal amounts of protein extracts were subjected to SDS/polyacrylamide gel electrophoresis under reducing conditions on concentrate protein gel 5% (pH = 6.8) and separating protein gel 12% (pH = 8.8). The separated proteins were transferred to PVDF membranes using a tank transfer for 2 h at 200 mA in Tris-glycine buffer with 15% methanol. Membranes were blocked with 5% skimmed milk for 3 h and incubated for 12 h with anti-CYP2E1 (1:1500, BOSTER, Wuhan, China), anti-Nrf-2 (1:500, Bioss, Beijing, China), anti-GAPDH (1:1000, BOSTER, Wuhan, China) and anti-Lamin B (1:500, Bioss, Beijing, China) for 2 h at 37 °C. The secondary antibodies (IgG/HRP) were incubated for 2 h at 37°C. The images of the blots were visualized by ECL (Genshare, Xi’an, China).
7
Oxidative Stress Mitigation in Cells
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OTA (purity > 98%) was provided by Pribolab (Immunos, Singapore); Se-Y (contained Se 2000 mg/kg) was obtained from Angel Yeast (Beijing, China) and was dissolved in autoclaved saline eluent. Kits for analyzing ALT, AST, MDA, GSH-Px, SOD and T-AOC contents were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total tissue RNA extraction kit, cDNA synthesis kit and qRT-PCR kit were acquired from Vazyme (Nanjing, China). Anti-Caspase3 (1:900, Abcam, Tokyo, Japan), anti-Bax (1:900, Immunoway, Suzhou, China), anti-HO-1, anti-Bcl-2, anti-Nrf2 (1:800, Bioss, Beijing, China), anti-MnSOD (1:6000, Enzo, Farmingdale, NY, USA), anti-actin (1:10,000, Abcam, Tokyo, Japan) and the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:12,000, Jackson Immuno, PA, USA).
8
Quantifying Nrf2 and CYP2E1 Protein Expression
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For Nrf2 expression analysis, the extraction and isolation of cytoplasmic and nuclear proteins were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China), according to the manufacturer’s instructions. For CYP2E1 expression analysis, the extraction and isolation of microsomal proteins were carried out as described previously (Jiang et al., 2016 (link); Chen et al., 2019 (link)). The protein concentration was determined by BCA assay kit (Beyotime, Nanjing, China). Equal amounts of protein extracts were subjected to SDS–polyacrylamide gel electrophoresis under reducing conditions in concentrate protein gel 5% (pH = 6.8) and separating protein gel 12% (pH = 8.8). The separated proteins were transferred to PVDF membranes using tank transfer for 2 h at 200 mA in Tris–glycine buffer with 15% methanol. Membranes were blocked with 5% skimmed milk for 3 h and incubated for 12 h with anti-CYP2E1 (1:1500, Boster, Wuhan, China), anti-Nrf-2 (1:500, Bioss, Beijing, China), anti-GAPDH (1:1000, Boster, Wuhan, China), and anti-Lamin B (1:500, Bioss, Beijing, China) for 2 h at 37°C. The secondary antibodies (IgG/HRP) were incubated for 2 h at 37°C. The images of the blots were visualized by ECL (Genshare, Xi’an, China).
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