Western blots were performed according to our previous studies.19 , 20 A Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology) was used for the protein extraction. Primary antibodies against Nrf2 (Abcam), keap1 (Abcam), heme oxygenase‐1 (Abcam), SOD2 (Abcam), NOX2/gp91phox (Abcam), eNOS, eNOS (P‐S1177) (Abcam), GAPDH (Cell Signaling Technology) and lamin B (Abcam) were used. Goat anti‐rabbit IgG (LI‐COR) or goat antimouse IgG (LI‐COR) was used as the secondary antibody.
Heme oxygenase 1
Manufactured by Abcam
Sourced in United States, United Kingdom
Heme oxygenase-1 is an enzyme that catalyzes the degradation of heme, a vital component of various proteins. It plays a crucial role in maintaining cellular homeostasis and regulating oxidative stress response.
Automatically generated - may contain errors
Lab products found in correlation
Lamin b, by Abcam (2 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)
Keap1, by Abcam (1 mentions)
Goat anti rabbit igg, by LI COR (1 mentions)
Nox2 gp91phox, by Abcam (1 mentions)
Goat anti mouse igg, by LI COR (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl detection system, by Bio-Rad (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Absin (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Abcam (1 mentions)
Anti rabbit secondary antibody, by Proteintech (1 mentions)
Cox 2, by Abcam (1 mentions)
Protease phosphatase inhibitor, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
6 protocols using heme oxygenase 1
1
Western Blot Analysis of Antioxidant Proteins
2
Quantitative Western Blot Analysis
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Western blotting was performed as described by our laboratory [21 (link),22 (link)]. Briefly, cells after respective treatment were washed with phosphate buffered saline (PBS) and whole cell lysates were collected in radioimmunoprecipitation (RIPA) buffer containing protease inhibitor cocktail, and phenyl methane sulfonyl fluoride (PMSF). Cell lysates were sonicated, centrifuged for 5 min at 14,000 rpm at 4°C, and protein concentrations were quantified by BCA protein assay kit (Pierce, Rockford, IL). Thirty micrograms of protein was resolved on a 4–20% sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. Blots were incubated overnight at 4°C in respective primary antibody [Nrf2 (1: 250) Santa Cruz Biotechnology, Santa Cruz, CA), heme oxygenase-1 (1: 1000; Abcam, Cambridge, MA), or β-actin (1:3000; Sigma, St Louis, MO)] followed by a 2-h incubation with a corresponding Alexa Fluor secondary antibody. Blots were visualized using the Li-Cor Odyssey near-infrared imaging system and quantified using Quantity One software (Bio-Rad, Foster City, CA).
3
Cardiac Tissue Protein Extraction and Analysis
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We extracted cytoplasmic proteins and nuclear proteins from cardiac tissue samples according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Absin, Shanghai, China) and quantified for protein levels using a BCA assay kit (Thermo Fisher, Shanghai, China). The specific process has been previously described [16 (link)]. A total of 50 μg protein from heart homogenates was applied per lane for separation by SDS-PAGE. Separated protein was immunoblotted and probed with the following primary antibodies overnight at 4°C: NF-E2-related factor 2 (Nrf2; 1:1000), heme oxygenase-1 (HO-1; 1:1000), Lamin B (1:1000), MR-1 (1:1000), and β-actin (1:1000), all from Abcam, Cambridge, MA, USA. HRP-conjugated secondary antibodies (1:3000, Abcam) were used to label the proteins. Subsequently, we then visualized the bands of target protein using an ECL detection system (Bio-Rad). The information of proteins was normalized to β-actin or Lamin B signals.
4
Protein Immunoblot Analysis of Retinal Ganglion Cells
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An automated protein immunoblot was performed using Simple Wes Wb (ProteinSimple, San Jose, CA) for protein detection. After treatment, RGCs were washed with ice-cold PBS, scraped, and then incubated in lysis buffer containing RIPA (Beyotime Institute of Biotechnology, Shanghai, China), supplemented with protease phosphatase inhibitors (Roche Pharmaceutical Ltd., Basel, Switzerland) on ice for 10 min. The lysate was centrifuged at 6000 rpm for 5 min at 4 °C and the supernatant was collected. Total protein concentration of the supernatant was determined by BCA microplate assay (Pierce BCA Protein Assay Kit) according to manufacturer’s instructions. The protein concentration of each sample was adjusted to 0.5 μg/μl and the sample loaded according to manufacturer’s instructions. Primary antibodies are as follows: iNOS (Genetex, USA), COX-2 (abcam, UK), Nrf-2 (abcam, UK), heme oxygenase-1 (abcam, UK), GPx-1/2 (Santa, USA), Akt (pS472/pS473) (BD PMG, USA), Bcl-2 (BD PMG, USA), Bax (BD PMG, USA), caspase-3 (CST, USA), GAPDH (abcam, UK)*, anti-rabbit secondary antibody (Proteintech, PRC). All the primary antibodies were diluted 1:50 and secondary antibody diluted 1:100. The proteins were quantified and analyzed with Compass software (ProteinSimple, San Jose, California, USA). Values of each index were normalized to GAPDH to calculate relative protein expression levels.
5
Western Blot and Immunohistochemistry Protocols
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The following antibodies (Abcam, Inc., Cambridge, MA) were used for western blotting: heme oxygenase-1 (rabbit monoclonal, clone EP1391Y), Nrf2 (rabbit monoclonal, clone EP1808Y), CD31 (rabbit monoclonal, clone EPR3094). Secondary antibody used for western blotting was polyclonal goat anti-rabbit (H+L), conjugated to horseradish peroxidase (Life Technologies, cat. # G-21234). The following antibody was used for immunohistochemical staining of Nrf2 in cells [66 (link)]: Nrf2 (H-300), sc-13032 rabbit polyclonal raised against amino acids 37–336 of human Nrf2 (Santa Cruz Biotechnology Inc.). Secondary antibody used for immunohistochemical staining was goat anti-rabbit IgG (H+L) Alexa Fluor 488 conjugate (ThermoFisher).
6
Antibody Usage for Cellular Analysis
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