After pretreatment with CSN-B and/or MPP+ and/or siNur77, the cells were rinsed thrice with ice-cold PBS and incubated in ice-cold lysis buffer for 20 min on ice. The supernatants were then collected and subjected to a bicinchoninic acid assay to determine the protein concentrations. The samples were then mixed with 5× Loading buffer and heated for 5 min at 100ºC. Next, aliquots with equivalent amounts of total protein (30 μg) were separated on NuPageBis-Tris 10% gels (Invitrogen, USA) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4ºC in solutions with primary antibodies (1:2000) against TNF-α, IL-6, MCP-1(Abcam, Cambridge, UK), and (1:2000) against Nrf2, HO-1, NQO-1, NF-κB p65 and Phospho IκB-α (Cell Signaling Technology, Danvers, MA), and primary antibodies (1:5000) against β-Actin (CWBio, Beijing, China), washed thrice and incubated with horse radish peroxidase-conjugated secondary antibodies. Negative controls were prepared by excluding the primary antibodies. Images of the labeled membranes were analyzed using Image J software (National Institutes of Health, USA).
Nupagebis tris 10 gels
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPageBis-Tris 10% gels are pre-cast polyacrylamide gel electrophoresis (PAGE) products designed for the separation and analysis of proteins. These gels utilize a Bis-Tris buffer system and have a concentration of 10% acrylamide. The gels are intended for use in standard vertical gel electrophoresis systems.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay reagent, by Bio-Rad (3 mentions)
Histone extraction kit, by Abcam (3 mentions)
Pvdf membrane, by GE Healthcare (3 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Ecl antirabbit igg horseradish peroxidase linked whole antibody, by GE Healthcare (2 mentions)
Tnf α, by Abcam (1 mentions)
β actin, by CWBIO (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Mcp 1, by Abcam (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Nitrocellulose sheets, by Cytiva (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
Lowry method, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Kodak (1 mentions)
Ab6721, by Abcam (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
8 protocols using nupagebis tris 10 gels
1
Protein Expression Analysis in Treated Cells
2
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were harvested using cell scrapers, washed in ice-cold PBS, and lysed with two different ice-cold lysis buffers. The supernatants were collected for protein determination by BCA assay (Pierce, Inc., Rockford, IL, USA), and the proteins were run on NuPage Bis-Tris 10% gels (Invitrogen, Waltham, MA, USA) and transferred to PVDF membranes (Amersham Bioscience, Ltd., Buckinghamshire, UK). The membranes were blocked in 5% skim milk, 0.05% Tween 20, and Tris-buffered saline (TBS) for 1 h. The PVDF membranes were incubated in the primary antibodies overnight at 4 °C. For detailed antibody information, please refer to the Supplementary Information . The next day, horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, Danvers, MA, USA) were applied. Peroxidase-conjugated streptavidin and substrate were used for detection. Negative controls were prepared by omitting the primary antibodies. For the protein extractions prepared from the cytosolic and nuclear fractions, the method described by Garcia-Yagüe et al.59 (link) was used. The images were analyzed using NIH Image J software (Bethesda, MD, USA). For additional details, please refer to the Supplementary Information .
3
Protein Expression Analysis in HRMECs and Retina
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HRMECs and retina proteins were collected. NuPAGE Bis-Tris (10%) gels (Invitrogen) were used according to the manufacturer’s instructions. The membranes were blocked with 10% fat-free milk and incubated with primary antibodies overnight at 4 °C. The primary antibodies were used at the following dilutions: anti-Akt (rabbit monoclonal, CST#9272, 1:1000), phospho-Akt (Thr308; rabbit monoclonal, CST#13038, 1:1000), phospho-Akt (Ser473; rabbit monoclonal, CST#4060, 1:1000), phospho-Akt (Thr450; rabbit monoclonal, CST#12178, 1:1000), and anti-SHIP1 (D1163; rabbit monoclonal, CST#2728, 1:1000). To control for lane loading, the same membranes were probed with anti-beta-actin (mouse monoclonal antibody, ab8226, 1:2000) after being washed with a stripping buffer. Relative changes in protein expression were calculated and expressed as fold changes. Each experiment was repeated at least three times.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total fraction of proteins was extracted from uteri by adding 400 μL of RIPA buffer (Tris-HCl pH 7.6, NaCl 150 mM, EDTA 2 mM, Glycerol 10%, Triton-X100 1%, sodium deoxycholate 0.5% and sodium lauryl sulfate 0.2%). Phenylmethylsulfonyl fluoride (Sigma, Saint Louis, MO, USA) and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) were also added. Proteins were quantitated by Lowry method (Bio-Rad Laboratories, Hercules, CA, USA). Samples were boiled and diluted 1:5 in Laemmli buffer (Bio-Rad Laboratories) and 2-mercaptoethanol (Bio-Rad Laboratories). 50 μg of total protein per sample were loaded on NuPAGE BIS-TRIS 10% gels (Invitrogen) and transferred to nitrocellulose sheets (Amersham Biosciences, Buckinghamshire, UK). Nonspecific binding was blocked with 5% non-fat dry milk in TBS-Tween 20 (0.5%) for 1 h at room temperature. Primary antibodies: monoclonal rabbit anti–IL-6 (1:1,000) D5W4V clone (Cell Signaling Technology, Danvers, MA, USA); polyclonal rabbit anti–NFKBID (IκBNS) (1:500) ab182633 (Abcam, Cambridge, MA, USA). Secondary antibody: goat anti-rabbit IgG–Peroxidase (1:5000) ab6721 (Abcam, Cambridge, MA, USA). Chemiluminescence was developed with Pierce ECL Western blotting substrate and detected on blue autoradiography film (Kodak, Rochester, NY).
5
Histone Protein Extraction and Western Blot
6
Western Blot Analysis of Brain Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen whole-brain tissue was minced and homogenized with a Polytron homogenizer in ice-cold PBS (five times the brain weight) containing phenylmethylsulfonyl fluoride. The samples were then sonicated. Before Western blot analysis, protein concentrations were determined using the Lowry method. We adjusted the protein concentration to 2 mg/mL and loaded 10 μg of the total protein in each well. Proteins were separated by NuPAGE Bis-Tris 10% gels (Thermo Fisher) and transferred onto PVDF membranes (Millipore). The membranes were blocked with Blocking One (Nacalai) for 1 h, followed by overnight incubation at 4 °C with primary antibodies in the blocking solution. Primary antibodies for iNOS (mouse monoclonal IgG (Pharmingen)), Arginase-1 (rabbit polyclonal IgG (Gene Tex)), and α-Tubulin (mouse monoclonal (Novus Biologicals)) were used. After 1-hour incubation in a horseradish peroxidase-conjugated secondary antibody, immunoreactive bands were visualized by enhanced chemiluminescence (ECL prime, GE Healthcare) and ImageQuant LAS4000 mini (GE Healthcare). Intensities of the bands of interest were quantified using Image J software.
7
Western Blot Analysis of Protein Extracts
8
Histone Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Histone lysates were extracted using the Histone Extraction Kit (Abcam). Lysate protein concentration was determined with the Bradford assay reagent (Bio-Rad). Three micrograms of histone was separated on NuPAGE Bis-Tris 10% gels (ThermoFischer Scientific) and wet-transferred to a PVDF membrane (GE Healthcare). Membrane blocking was performed with 5% skim milk in Tris-buffered saline (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.4) (TBST) for 1 hour. Membranes were incubated overnight with primary antibody in 1% skim milk in TBST. Membranes were washed 3 times in TBST, and the secondary antibody (ECL anti-rabbit IgG Horseradish Peroxidase linked whole antibody) (GE Healthcare) was applied for 1 hour in 1% skim milk in TBST. Membranes were washed 3 times and the signal was resolved with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and imaged on a ChemiDoc MP Imaging System (Bio-Rad). The antibodies and their concentrations are listed in the Life Sciences Reporting Summary.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!