Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology) was used to extract proteins and the concentrations were determined with Bicinchoninic Acid (BCA) assay (Bio-Rad Laboratories, Inc.). Equivalent amounts of proteins (40 µg) were separated by 10% SDS-PAGE electrophoresis and later moved onto polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked at room temperature with 5% skimmed milk for 1 h. Incubation was performed overnight using appropriated primary antibodies (rabbit polyclonal antibodies for TGF-βR1 cat. no. 41896S; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat. no. ab5694; 1:2,000; Abcam) at 4°C. Afterward, the PVDF membranes in horseradish peroxidase-conjugated secondary antibodies (cat. no. ab6721, 1:2,000; Abcam) was incubated for 2 h at room temperature. Protein blots were visualized with the ECL-Plus Western Blot Analysis Detection System (Thermo Fisher Scientific, Inc.) and the band densities were quantified using ImageJ software (V1.8.0.112; National Institutes of Health).
Ecl plus western blot analysis detection system
Manufactured by Thermo Fisher Scientific
The ECL-Plus Western Blot Analysis Detection System is a chemiluminescent detection system designed for western blot analysis. It provides a sensitive and quantitative method for detecting and analyzing specific proteins in complex samples.
Automatically generated - may contain errors
2 protocols using ecl plus western blot analysis detection system
1
Western Blot Analysis of TGF-βR1 Expression
2
Western Blot Analysis of Prostate Cancer
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Proteins of tumor tissues from PC patients or nude mice, PC cells (PC-3 and LNCap) lysates were harvested with RIPA lysis buffer (Sangon Biotech, Shanghai, China) supplemented with protease inhibitor. Protein concentration was determined using the BCA protein assay. Then 25 μg of protein from each sample was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA) in transfer buffer. After transfer, the membrane was blocked with 5% non-fat milk for 1 h at room temperature, and incubated with the following primary antibodies (BioTeke, Beijing, China) at 4 °C overnight: RAB11A (1:1000), N-cadherin (1:1000), E-cadherin (1:1000), Vimentin (1:1000), Snail (1:1000), β-catenin (1:1000), Cyclin D (1:1000), c-Myc (1:1000) and GAPDH (1:3000). Thereafter, the membranes were incubated with horseradish peroxidase-labelled secondary antibodies IgG (1:1000; BioTeke, Beijing, China) for 2 h at room temperature. Protein blots were visualized by using the ECL-Plus Western Blot Analysis Detection System (Thermo Fisher Scientific). GAPDH was used as an internal reference.
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