Cells were harvested by trypsinization and centrifugation. Total protein was extracted, and western blot analysis was performed as reported previously [20 (link)]. 50 μg of total protein extracted from cells after 72 h treatments was applied to a 10% polyacrylamide gel, and PageRuler Prestained Protein Ladder (Fermentas) was used as the size marker. After the proteins were transferred to membranes and the membranes were blocked, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies used in this study included Rabbit anti-human E-Cadherin (0.3 μg/ml), N-Cadherin(1 μg/ml), Vimentin(0.2 μg/ml), Notch1(0.85 μg/ml), Numb(0.26 μg/ml), Slug(0.25 μg/ml), Snail(0.2 μg/ml) and β-actin (0.5 μg/ml). After incubation with the Goat anti-rabbit HRP-conjugated secondary antibody (0.1 μg/ml) for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). β-actin was used as the loading control.
Ecl detection system
Manufactured by BD
Sourced in United States
The ECL detection system is a laboratory equipment used for the detection and analysis of chemiluminescent signals. It functions by capturing and quantifying the light output from chemiluminescent reactions, providing a sensitive and reliable method for various applications in the field of biochemistry and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Anti human e cadherin, by Cell Signaling Technology (1 mentions)
Anti human vimentin, by Cell Signaling Technology (1 mentions)
Anti human phospho p70s6k, by Cell Signaling Technology (1 mentions)
Anti human stat3, by Cell Signaling Technology (1 mentions)
Nitrocellulose filter, by Merck Group (1 mentions)
7 protocols using ecl detection system
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of EMT Markers
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The cells were harvested by centrifugation and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Polyvinylidene Fluoride (PVDF) membranes by electroblotting. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. The anti-human E-cadherin, anti-human vimentin, anti-human SNAILl2, anti-human STAT3, and anti-human phospho-p70s6k primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-human β-actin primary antibodies and Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China). After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences, New York, NY, USA).
3
Western Blot Protein Analysis
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Cell lysates were prepared using previously described methods [31 (link)]. Equal amounts of cell lysate were separated using SDS-PAGE, electroblotted onto a nitrocellulose filter (Millipore), and analyzed using the specified antibodies with an ECL detection system (BD Biosciences, San Diego, CA, USA).
4
Western Blot Protein Analysis Protocol
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The cells were harvested by centrifugation and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of the protein lysates were electrophoretically separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies. After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). β-actin or GAPDH were used as the loading controls.
5
Western Blot Analysis of Protein Expression
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The cells (1×107) were harvested by centrifugation (1,000 rpm for 5 min) and washed with PBS. The cells were lysed in RIPA buffer containing protease inhibitors. Equal amounts of the protein lysates were electrophoretically separated on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline/0.1% Tween-20 for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies. After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). β-actin or GAPDH was used as the loading control. The experiment was performed 3 times.
6
Protein Expression Analysis of Cell Samples
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The cells were harvested by centrifugation and washed with PBS, and then they were lysed in RIPA buffer containing protease inhibitors. Equal amounts of the protein lysates were electrophoretically separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk and then incubated overnight at 4°C with the primary antibodies: Sanil2, STAT3, phosphor-p70s6k, E-cadherin, Vimentin, and β-actin, which were purchased from CST. After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). β-actin was used as the loading control.
7
Comparative Analysis of Western Blotting Techniques
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Simple Western blot analysis (ProteinSimple, California, USA) was performed by Simple Western system according to manufacturer’s guideline (https://www.proteinsimple.com.cn/wes.html ). Briefly, the proteins were diluted to a final protein concentration of 1–3 µg/µL in a master mix containing internal fluorescent standards and DTT. The primary antibodies against N-cadherin, NOTCH1, Slug, Vimentin, E-cadherin, Snail and β-actin that diluted to 1:20–1:10 were used. Quantification of protein expression was determined automatically by the software in the system.
For traditional western blot analysis, the cells were lysed in RIPA buffer containing protease inhibitors. Forty µg of the protein lysates were electrophoretically separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). GAPDH was used as the loading controls.
For traditional western blot analysis, the cells were lysed in RIPA buffer containing protease inhibitors. Forty µg of the protein lysates were electrophoretically separated on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffered saline/0.1% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies. After incubation with the secondary antibody for 1 h at room temperature, the protein bands were detected using the ECL detection system (BD Biosciences). GAPDH was used as the loading controls.
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