At 48 h after transfection, 106 transfected CNE2 cells were lysed with RIPA buffer (Beyotime, Jiangsu, China) then boiled for 5 min. Protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk in 1 × Tris buffered saline/0.1% Tween-20 (pH 7.4; TBST) at room temperature for 1 h, membranes were incubated overnight at 4℃ with rabbit antihuman antibodies to E-cadherin, Snail, caspase 9, Bcl-2, Bax, AKT and PTEN (all 1:500 dilution; Cell Signaling Technology, Beverly, MA, USA), washed three times with TBST for 10 min at room temperature, then incubated with horseradish peroxidase-conjugated mouse antirabbit secondary antibody (1:1000 dilution; Cell Signaling Technology, USA) for 1 h at room temperature. Membranes were washed three times with TBST for 10 min at room temperature and immunoreactive signals were visualized using an EasySee® Western Blot Kit (TransGen Biotech, Beijing, China). Protein bands were quantified by densitometry (Tanon-1600 gel image system; BioTanon, Shanghai, China).
Horseradish peroxidase conjugated mouse anti rabbit secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody is a reagent used in immunoassays and immunoblotting techniques. It consists of a mouse-derived antibody that binds to rabbit primary antibodies, conjugated with the enzyme horseradish peroxidase.
Automatically generated - may contain errors
Lab products found in correlation
Erα erβ, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Easysee western blot kit, by Transgene (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using horseradish peroxidase conjugated mouse anti rabbit secondary antibody
1
Western Blot Analysis of EMT and Apoptosis Markers
2
Quantitative Analysis of ER Protein Levels
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After the treatment cells were lysed with M-PER Mammalian Protein Extraction Reagent with Halt proteases inhibitor (ThermoFisher Scientific, Waltham, MA, USA). Equal aliquots (20 µg) of the protein samples were separated by 4–12% SDS-PAGE, transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA) and blocked with 5% BSA in TBST buffer. Membranes were incubated with rabbit β-actin (Cell Signaling Technology, Danvers, MA, USA) and ERα/ERβ (ThermoFisher Scientific) antibodies at 4 °C overnight, after which they were incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (Cell Signaling) for 1 h at room temperature. Western blots were developed using SuperSignal™ West Pico Chemiluminescent Substrate (ThermoFisher Scientific) and quantified by densitometry.
3
Western Blot Analysis of Estrogen Receptor Levels
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Cells were seeded into a 6-well plate at a density of 2 × 105 cells/well. After cell incubation for 48 h with extracts, the cells were lysed with M-PER Mammalian Protein Extraction Reagent with Halt protease inhibitor (ThermoFisher Scientific, Waltham, MA, USA). Equal aliquots (20 μg) of the protein samples were separated by 4–12% SDS-PAGE, transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA) and blocked with 5% BSA in TBST buffer. The membranes were incubated with rabbit β-actin (Cell Signaling) and ERα/ERβ (ThermoFisher Scientific) antibodies at 4 °C overnight, and then incubated with horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. The Western blots were developed using SuperSignal™ West Pico Chemiluminescent Substrate (ThermoFisher Scientific) and quantified by densitometry.
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