Liver tissues and L02 cells were homogenized in ice-cold RIPA Lysis Buffer (Beyotime, China) with PMSF. Concentrations of protein samples were detected by BCA protein assay (Beyotime, China). Western blotting was performed as previously described [27 (link)]. Protein samples with equal amounts were separated using SDS/PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% BSA for 1 h, membranes were incubated with specific primary antibodies overnight at 4 °C. The primary antibodies were shown as follows: Anti-GPX4 (Abways, CY6959, 1:1500), Anti-Ferritin (Abcam, ab75973, 1:2000), Anti-SLC7A11 (Abcam, ab175186, 1:2000), Anti-Nrf2 for L02 cells (Abcam, ab62352, 1:1000), Anti-Nrf2 for liver tissues (Abclonal, A11159, 1:1000), Anti-pNrf2 (Abcam, ab76026, 1:2000), Anti-GCLC (Abcam, ab20777, 1:1000), Anti-HO-1 (Abcam, ab189491, 1:1000), Anti-NQO1 (Abcam, 1:2000, ab28947), Anti-GAPDH (Abcam, ab181602, 1:10000). Then the membranes were washed with PBST for 3 times and incubated with Anti-rabbit and Anti-mouse IgG HPR-linked secondary antibodies for 1 h, followed by chemiluminescence detection using ChemiDocTM Touch Imaging System (BIO-RAD, California, USA).
Ab189491
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab189491 is a monoclonal antibody that can be used as a research tool. It is designed to detect a specific target protein. The antibody is suitable for use in various laboratory techniques, such as immunohistochemistry, western blotting, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Ab125066, by Abcam (4 mentions)
Ab75973, by Abcam (3 mentions)
Ab92946, by Abcam (3 mentions)
Ab8227, by Abcam (3 mentions)
Ab137550, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab181602, by Abcam (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
Ab155282, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab76026, by Abcam (1 mentions)
Ab28947, by Abcam (1 mentions)
Ab175186, by Abcam (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Ab13867, by Abcam (1 mentions)
20 protocols using ab189491
1
Western Blot Analysis of Antioxidant Proteins
2
Retinal Protein Extraction and Western Blot
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Total protein extracted from pulverized retinal samples was mixed with lysis buffer (radioimmunoprecipitation assay buffer containing 1% phenylmethylsulfonyl fluoride). The concentration of the extracted protein was quantified with the bicinchoninic acid protein quantification kit (Beyotime Biotechnology, Shanghai, China). The protein samples were denatured by heating, electrophoresed on 10 or 12% SDS-PAGE gels, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk, and then incubated with primary antibodies (anti-HO-1 (1:500; rabbit monoclonal; Cat# ab189491, Abcam, Cambridge, UK), anti-NLRP3 (1:500; rabbit monoclonal; Cat# ab263899, Abcam), anti-TLR4 (1:500; rabbit polyclonal; Cat# ab13867, Abcam), anti-ASC (1:500; rabbit polyclonal; Cat# bs-6741R, Bioss, Beijing, China) or anti-caspase-1 (1:1000; rabbit polyclonal; Cat# bs-0169R, Bioss)) at 4°C overnight. Then, the blots were incubated with secondary antibodies labeled with horseradish peroxidase (1:10,000; ZSGB-BIO, Beijing, China), and thereafter subjected to chemiluminescent detection. Signal intensities were analyzed using ImageJ software and normalized to the normal control.
3
Western Blot Analysis of Protein Levels
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Equal amounts of protein were separated by SDS-PAGE and then incubated overnight with specific antibodies against HMOX1(1:2000)(Abcam, ab189491)/FTH(1:1500) (Abcam, ab75973)/GAPDH(1:20000)(Affinity, AF7021). After washing, the blots were incubated with a secondary antibody(1:10000) (Immunoway, RS0002). The data were analyzed using ImageJ software.
4
Immunofluorescent Staining of Cellular Proteins
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The cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 10% goat serum (Beyotime, C0265) for 1 h at room temperature, incubated with HMOX1(1:250)(Abcam, ab189491)/FTH(1:100) (Abcam, ab75973) antibody at 4 °C overnight, then added FITC IgG (H + L)(1:1000)(Immunoway, RS0004) at room temperature for 1 h. Finally, we added DAPI to stain the cell nucleus. Images were acquired with a fluorescence microscope (Olympus, Japan) using DP Manager software.
5
Nrf2-HO-1 Pathway in Iron Homeostasis
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The treated TG905 cells were washed thrice with ice-cold PBS and homogenized with RIPA lysate containing 1 mM phenylmethanesulfonyl fluoride. Samples were centrifuged at 12000 rpm for 10 min, and the supernatant were collected. The total protein concentration was measured using BCA protein assay kits (Solabio). Cell lysates were separated using 10% SDS-PAGE and electro-transferred onto a polyvinylidene difluoride membrane. After incubation in blocking buffer (1×TBS, 0.1% Tween-20 and 5% w/v dry nonfat milk) at 4℃, membranes were incubated with the desired primary antibody, followed by incubation with the appropriate peroxidase-conjugated secondary antibody. The primary antibodies included those for Nrf2 (16396-1-AP, Proteintech, Wuhan, China), HO-1 (ab189491, Abcam, Cambridge, England), DMT1 (20507-1-AP, Proteintech), GPX4 (ab125006, Abcam), ferritin (ab75973, Abcam), transferrin receptor (ab214039, Abcam), ferroportin-1 (bs4906R, Bioss, Beijing, China), and GAPDH(ab181602, Abcam). The films were scanned, and the intensity values of each band were determined using an Alphalmager HP system (Cell Biosciences Inc., Santa Clara, CA, USA).
6
Western Blot Analysis of Lung Tissue
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Lung tissue samples or cells were lysed in a RIPA buffer (Beyotime, P0013B, China) with protease inhibitor cocktail (Roche, 4693132001, USA) for 20 min. The protein concentrations were measured using a BCA protein assay kit (Beyotime, P0010, China), and 50 μg of proteins were transferred onto a PVDF membrane following separation on a 10% SDS-polyacrylamide gel. The membrane was blocked with blocking solution (5% (w/v) nonfat dry milk) for 2 h, followed by an overnight incubation at 4 °C with antibodies against KEAP1 (Cell Signaling Technology, 8047, 1:1000), NRF2 (Cell Signaling Technology, 12,721, 1:1000), GSTP (Cell Signaling Technology, 3369, 1:1000), HO-1 (Abcam, ab189491, 1:2000), NQO1 (Abcam, ab80588, 1:10,000), Lamin B (Abcam, ab32535, 1:500) and β-Actin (Cell Signaling Technology, 8457, 1:1000). The following day, the membrane was incubated for an additional 1 h with HRP-conjugated secondary antibody (1:1000 dilution) at room temperature after thoroughly washing three times with PBST. Bands were detected by ECL (Beyotime, P0018AM, China) and band intensities were quantified using Image J gel analysis software. All experiments were performed in triplicate.
7
Osteoclastogenesis Regulation via Nanoparticles
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Ti nanoparticles were obtained from Nanjing Emperor Nano Materials Company. RNA interference sequences were purchased from GenePharma (Suzhou, China). Murine RANKL was purchased from R&D Systems (Minneapolis, USA). GKT137831 was purchased from ApexBio (Boston, USA). Dulbecco’s modified Eagle’s medium (DMEM/high glucose) was purchased from VivaCell (Shanghai, China) and fetal bovine serum (FBS) was obtained from HyClone (Logan, USA). CCK-8 assay kits were obtained from ApexBio (Boston, USA). The primary antibodies used in our study included NFATc1 (A1539, ABclonal, Wuhan, China), MMP-9 (A0289, ABclonal), NOX4 (A11274, ABclonal), HO-1 (ab189491, Abcam Cambridge, UK), SOD2 (ab137037, Abcam), and Nrf2 (A0674, ABclonal). Secondary antibody was purchased from Multisciences (Hangzhou, China).
8
Western Blot Analysis of Protein Expression
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Total protein was extracted from H9C2 cells using the total protein extraction kit (Applygen Technologies, Beijing, China), and protein concentration was examined using bicinchoninic acid kit (Beyotime). An equal amount of protein (30 μg) was separated by 12% SDS/PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h and incubated with the primary antibodies GPX4 (1 : 1000, ab125066; Abcam, Cambridge, MA, USA), ACSL4 (1 : 10 000, ab155282; Abcam), Nrf2 (1 : 1000, ab92946; Abcam), HO‐1 (1 : 2000, ab189491; Abcam) and β‐actin (1 : 1000, ab8227; Abcam) at 4 °C overnight. Afterward, the membranes were incubated with horseradish peroxidase‐labeled secondary antibody (1 : 2000, ab205718; Abcam) for 1 h. The protein band was visualized using enhanced chemiluminescence system (Thermo Fisher Scientific). The band intensity was analyzed using imagej software (NIH Image, Bethesda, MD, USA).
9
Quantitative Western Blot Analysis of Kidney Proteins
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Total protein was isolated from kidney cortex tissues using Radio Immunoprecipitation Assay Lysis buffer with protease phosphatase inhibitors and protease inhibitors obtained from Beyotime, and the extract was centrifuged at 10,000 ×g for 15 min to remove cell debris. Protein concentration was quantified using a BCA protein assay kit (Beyotime). Then, protein samples (20 μg/group) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane. After blocking with 5% skimmed milk, the membrane was incubated overnight with primary antibodies against Bax (ab182733, 1:2000; Abcam), Nrf2 (#20733, 1:1000; Cell Signaling Technology), cleaved caspase-3 (#9661, 1:1000; Cell Signaling Technology), Sirt1 (ab189494, 1:1000; Abcam), Bcl-2 (ab196495, 1:2000; Abcam), β-actin (ab8227, 1:2000; Abcam), and HO-1 (ab189491, 1:2000; Abcam) at 4 °C. Then the membranes were incubated with secondary antibodies for 2 h at room temperature. The bands were visualized using an enhanced chemiluminescent reagent (Yeasen, Shanghai, China). The immunoblot images were evaluated by ImageJ software.
10
Protein Expression Analysis via Western Blot
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Western blot analysis to assess protein expression was performed as previously described [23 (link)]. Frozen hearts were mixed in RIPA buffer (with protease inhibitor), centrifuged (10,000 rpm, 8 min, 4 °C), and then the supernatant was assessed using a NanoDropLite spectrophotometer (Thermo Fisher Scientific, Austin, TX, USA) to determine the amount of protein. Protein samples (45 µg) were electrophoresed in SDS-PAGE, transported to the polyvinylidene fluoride (PDVF) membrane, incubated with 5% BSA and then with primary antibodies of Keap1 (ab139729), Nrf2 (ab92946), HO-1 (ab189491), PI3K (ab227204), p-PI3K (ab182651), Akt (ab210454), p-Akt (ab38449), mTOR (ab134903), p-mTOR (ab137133) and housekeeping anti-β actin (ab8227), which were obtained from Abcam (Boston, MA, USA) and diluted up to 1:1000. These primary antibodies were identified using horseradish peroxidase (HRP)-conjugated secondary antibodies. Antigen-antibody reactions were visualized with an enhanced chemiluminescence kit (ECL, Sigma-Aldrich) under a gel documentation system. Image J software (National Institutes of Health, University of Wisconsin, Madison, WI, USA) was used to evaluate the acquired images.
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