Potassium chloride (KCl), PE, Nω-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), ACh, sodium nitroprusside (SNP), 4-Hydroxy-TEMPO (tempol, a superoxide anion scavenger), indomethacin (a non-selective cyclooxygenase (COX) inhibitor), and methyl-β-cyclodextrin (CD, a cholesterol depleter which disassembles caveolae) were purchased from Sigma-Aldrich (St. Louis, MO, USA); losartan (an Ang II type 1 receptor antagonist) was from Cayman Chemical Company (Ann Arbor, MI, USA). Drug concentrations were expressed as final molar concentrations in the bath chamber. 10× radio-immunoprecipitation assay (RIPA) lysis buffer was from Millipore Corporate Headquarters (Temecula, CA, USA); bicinchoninic acid (BCA) protein assay kit and Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate were from Thermo Fisher Scientific (Rockford, IL, USA); anti-p-eNOSThr495 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-p-eNOSSer1177 was from Abcam plc (Cambridge, UK); anti-eNOS, anti-adenosine monophosphate-activated protein kinase (anti-AMPK), anti-Cav-1, anti-β-actin, and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG were from BD Transduction Laboratorie (Lexington, KY, USA); HRP conjugated goat anti-rabbit IgG was from Cell Signaling Technology (Danvers, MA, USA).
Pierce enhanced chemiluminescent ecl western blotting substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce enhanced chemiluminescent (ECL) Western Blotting Substrate is a laboratory reagent used in the detection and analysis of proteins through Western blotting techniques. It provides a chemiluminescent signal that can be detected and quantified using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Losartan, by Cayman Chemical (1 mentions)
Hrp conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
4 hydroxy tempo, by Merck Group (1 mentions)
Nω nitro l arginine methyl ester, by Merck Group (1 mentions)
Methyl β cyclodextrin cd, by Merck Group (1 mentions)
Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Sodium nitroprusside, by Merck Group (1 mentions)
Ab37168, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse, by Proteintech (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Proteintech (1 mentions)
Rabbit anti c ebpβ, by Santa Cruz Biotechnology (1 mentions)
Mouse anti tubulin, by Proteintech (1 mentions)
3 protocols using pierce enhanced chemiluminescent ecl western blotting substrate
1
Pharmacological Modulation of Endothelial Function
2
Western Blot Analysis of MAP2K1 Protein
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Protein samples were extracted from tumor tissues or cells by RIPA Lysis Buffer (Beyotime) and then 25 μg extracted proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After 2 h electrophoresis, the proteins in the gels were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Next, the membranes were blocked in 5% non-fat milk for 2 h and subsequently incubated overnight at 4°C with the primary antibodies of MAP2K1 (ab96653; Abcam, Cambridge, MA, USA) or GAPDH (ab37168, Abcam). GAPDH acted as the internal reference in this study. The next day, the membranes were probed with horseradish peroxidase (HRP)-conjugated second antibody (ab6721, Abcam). Finally, proteins bands were visualized using Pierce™ Enhanced chemiluminescent (ECL) Western Blotting Substrate (Thermo Fisher Scientific) with the intensity analysis via Quantity One software (Bio-Rad Laboratories).
3
Western Blotting of Tumor Cell Proteins
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Tumor cells or tumor tissues were lysed in radio immunoprecipitation assay buffer for Western blotting. The proteins were separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto polyvinylidene difluoride membranes (Millipore), which were then blocked in tris-buffered saline and Tween 20 (TBST) containing 5% defatted milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C with mouse anti-tubulin (1:10,000; Proteintech Group, Chicago, IL, USA) and rabbit anti-CEBPβ (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed with TBST and incubated with either horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5,000; Proteintech Group) or HRP-conjugated goat anti-mouse (1:5,000; Proteintech Group) secondary antibody, immune complexes were visualized by using Pierce™ enhanced chemiluminescent (ECL) Western Blotting Substrate (Thermo Fisher Scientific).
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