Cells were lysed in RIPA buffer (25 mmol/L Tris–HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% deoxycholate, 1% Triton X-100, 0.5% SDS, 2 mmol/L EDTA, 0.5 mmol/L PMSF, protease inhibitor cocktail). Protein was quantified with Bio-Rad protein assay reagents. Prestained molecular markers (Fermentas) were used to estimate the molecular weight of samples. Proteins were transferred to PVDF membranes (Millipore), and after incubation with primary and secondary antibodies, were detected by ECL regents (Amersham Biosciences). Bands were normalized to β-actin and expressed as a percent of control.
Ecl regent
Manufactured by Cytiva
Sourced in United States, United Kingdom
ECL reagents are a set of chemical solutions used in Western blotting techniques to detect and quantify target proteins. They facilitate the chemiluminescent detection of proteins that have been separated and transferred onto a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg conjugated to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (1 mentions)
Anti cdk4, by Abcam (1 mentions)
Anti dlx2, by Abcam (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Hrp labeled secondary antibody, by Abcam (1 mentions)
Anti cleaved parp, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ripa reagent, by Beyotime (1 mentions)
Anti cyclin d1, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
4 protocols using ecl regent
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Cells were washed and lysed in 0.15 ml cell extraction buffer (Invitrogen). Equivalent amounts of protein were solubilized in 2× SDS sample buffer, separated on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membrane, blocked with 5% nonfat dry milk containing 0.1% Tween 20 for 1 h at room temperature, and washed thrice with wash buffer (1×TBST). Blots were incubated with primary antibodies against AP-2α, α7 nAChR at 1:1000 dilution, or EP4 (1:4000), overnight at 4°C, then washed thoroughly, and incubated with secondary anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000 dilution; Santa Cruz) for 1 h at room temperature. Blots were stained by ECL regents (Amersham Life Science) and exposed to X-ray film, and proteins were quantified by densitometric scanning using a Bio-Rad GS-800 calibrated densitometer.
3
Western Blot Analysis of Protein Samples
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Cell lysates were prepared in RIPA buffer containing protease inhibitors as described [25 (link)]. In total, 60 μg of cell extracts were subjected to SDS-PAGE, transferred to PVDF membrane, and blotted with specific primary antibodies. For detection, membranes were incubated with secondary antibodies for 1 h, and results were visualized with ECL regent (ECL kit, Amersham Biosciences, Piscataway, NJ, USA), then quantified by Labscan software.
4
Western Blot Analysis of Cell Signaling Proteins
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Western blot assay was performed as described previously [21 (link)]. Tissues and cell lysates were prepared by using RIPA Reagent (Beyotime, Shanghai, China) and the concentration was measured by BCA kit (Thermo Fisher Scientific). Proteins were loaded on 10% SDS-PAGE and were transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were immersed in 5% skim milk powder for 2 h, and then incubated at 4 °C overnight with anti-CyclinD1 (1:200, Abcam, Cambridge, MA, USA), anti-CDK4 (1:2000, Abcam), anti-PARP (1:1000, Cell Signaling Technology, Shanghai, China), anti-Cleaved PARP (1:1000, Abcam), anti-Cleaved caspase-3 (1:500, Abcam), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-N-cadherin (1 µg/mL, Abcam), anti-Vimentin (1:1000, Abcam), anti-DLX2 (1:500, Abcam), Ki67 (1:1000, Abcam) or anti-GAPDH (1:2500, Abcam). Next, membranes were washed and probed with the HRP-labeled secondary antibody (Abcam) for 1 h at 37 °C. Membranes were visualized via the ECL regent (Amersham Biosciences, Buckinghamshire, UK).
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