Western blotting was performed, as previously described [14 (link)]. Briefly, hepatocytes were treated with 1 nM IL-1β and a fraction or constituent for 8 h and lysed in the presence of protease inhibitor cocktail (Nacalai Tesque, Inc.). The resultant lysates were run on a 10% sodium dodecyl sulfate–polyacrylamide gel and blotted onto a Sequi-Blot membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% skimmed milk, immunostaining was performed using primary antibodies against iNOS (Clone 54; BD Biosciences, San Jose, CA, USA) and β-tubulin (internal control; Cell Signaling Technology Inc., Danvers, MA, USA) and then horseradish peroxidase (HRP)-conjugated anti-immunoglobulin Fc antibody. The protein was visualized using enhanced chemiluminescence blotting detection reagents (GE Healthcare Biosciences Corp., Piscataway, NJ, USA) and detected using an Amersham Imager 600 (GE Healthcare).
Sequi blot membrane
Manufactured by Bio-Rad
Sourced in United States
The Sequi-Blot membrane is a nitrocellulose-based transfer membrane designed for use in Western blotting applications. It is used to immobilize proteins separated by gel electrophoresis for subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Amersham imager 600, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Rat β tubulin, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence blotting detection reagent, by Cytiva (2 mentions)
Nativepage running buffer, by Thermo Fisher Scientific (1 mentions)
Nativepage sample buffer, by Thermo Fisher Scientific (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Immune blot polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Nacalai Tesque (1 mentions)
Light blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Dark blue cathode buffer, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Goat anti mouse igg h l horseradish peroxidase hrp conjugate, by Bio-Rad (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Difco skim milk, by BD (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Nf κb inhibitor α iκb α, by Santa Cruz Biotechnology (1 mentions)
6 protocols using sequi blot membrane
1
Analyzing iNOS Expression in IL-1β-Treated Hepatocytes
2
Native PAGE Analysis of α2-Macroglobulin Purification
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Purified human α2-macroglobulin protein (725 kDa) was obtained from Enzo Life Science (New York, USA), anti-human monoclonal mouse α2-macroglobulin (immunoglobulin (Ig)G1 clone) from R&D Systems (Minneapolis, MN, USA), and goat anti-mouse IgG (H + L)-horseradish peroxidase (HRP) conjugate from Bio-Rad Laboratories (Hercules, CA, USA). Native PAGE™ Sample Buffer, Native PAGE Running Buffer, Dark Blue Cathode Buffer, Native Mark™ Unstained Protein Standard, and Light Blue Cathode Buffer were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DTT, Blocking One and Peroxidase Stain 3,3′-Diaminobenzidine kit (Brown Stain) were purchased from Nacalai Tesque (Kyoto, Japan). Immune-Blot® polyvinylidene difluoride membrane, Sequi-Blot™ membrane, Precision Plus Protein™ Dual Color Standards and Mini-PROTEAN® TGX™ were obtained from Bio-Rad Laboratories. Perfect NT Gel, Perfect NT Gel System and SDS-PAGE Running Buffer were obtained from DRC (Tokyo, Japan), and ECL Prime Western Blotting Detection Reagent from GE Healthcare (Buckinghamshire, UK).
3
Western Blot Analysis of iNOS Induction
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Western blotting was performed according to a previously described method [5 (link)]. Briefly, hepatocytes were incubated with 1 nM IL-1β ± fraction or compound for 8 h. Cell lysates were prepared in the presence of a protease inhibitor cocktail (Nacalai Tesque, Inc.). The resultant lysates were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a Sequi-Blot membrane (Bio-Rad, Hercules, CA, USA). After blocking with 5% Difco skim milk (BD Biosciences, San Jose, CA, USA), target proteins were immunostained using primary antibodies against iNOS (BD Biosciences) and β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by horseradish peroxidase-conjugated anti-immunoglobulin Fc antibody. The protein was visualized with Enhanced Chemiluminescence Blotting Detection Reagents (GE Healthcare Biosciences Corp., Piscataway, NJ, USA) and detected using an Amersham Imager 600 (GE Healthcare)
4
Hepatocyte Signaling Pathway Analysis
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Hepatocytes were treated with 0.1 nM IL-1β and 100 μg/ml FRLFE for 8 h on Day 1, and whole-cell lysates were prepared [23] (link). Briefly, hepatocytes (1×106 cells/35-mm dish) were lysed using sample buffer (125 mM Tris-HCl, pH 6.8, 5% glycerol, 2% sodium dodecyl sulfate (SDS), and 2% 2-mercaptoethanol), subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted onto a Sequi-Blot membrane (Bio-Rad, Hercules, CA, USA). Immunostaining was performed using primary antibodies that had been raised against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA), human NF-κB inhibitor α (IκB-α; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated IκB-α (Ser32/36 [5A5]), and rat β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by visualization with the Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).
5
GST Protein Immunoblotting and N-terminal Sequencing
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Purified proteins were separated by PAGE in the presence of 0.1% (w/v) of SDS with a polyacrylamide concentration of 10% (w/v), and then stained with Coomassie brilliant blue R250 (CBB or transferred to a polyvinylidene difluoride membrane (Merck-Millipore, Darmstadt, Germany). Immunoblotting was performed by using the anti-GST antiserum (2x10 3 -fold dilution), and blots were visualized with alkaline phosphatase-conjugated anti-rabbit Ig(G+A+M) using 5bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Promega). An anti-GST antiserum was developed with recombinant GST as an antigen in rabbits with the method reported previously (Ohara-Nemoto et al., 2011; 2014) . For the N-terminal sequencing, separated proteins were transferred to a Sequi-Blot membrane (Bio-Rad Laboratories, Hercules, CA), stained with CBB. The N-terminal sequences of CBB-stained bands were determined in Protein Research Institute (Osaka, Japan).
6
Immunoblotting of iNOS in Hepatocytes
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The hepatocytes were treated with 1 nM IL-1β and each constituent for 8 h, and whole-cell lysates were prepared [18] (link). Briefly, the hepatocytes were lysed, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted onto a Sequi-Blot membrane (Bio-Rad, Hercules, CA, USA). Immunostaining was performed using primary antibodies that were raised against rat iNOS (Thermo Fisher Scientific, Waltham, MA, USA) and rat β-tubulin (Cell Signaling Technology Inc., Danvers, MA, USA), followed by visualization with an Enhanced Chemiluminescence Blotting Detection Reagent (GE Healthcare Biosciences Corp., Piscataway, NJ, USA).
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