The glioma cells were harvested and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Beijing, China), and the protein concentration was measured with BCA Protein Assay kit (CWBio, Jiangsu, China). Equal amount of proteins were separated in 10% SDS/polyacrylamide gel, and then transferred on to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, U.S.A.). After blocking in 5% nonfat milk for 2 h at room temperature, membranes were incubated with primary antibodies: anti-E-cadherin, anti-Vimentin, or anti-β-actin (1:1000 dilution; Cell Signaling Technology, Beverly, MA, U.S.A.) at 4°C overnight. After washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 2 h at temperature. Then, immunoblots were visualized by enhanced chemiluminescence (CWBio, Jiangsu, China). Finally, Quantity One Software (Bio-Rad, Hercules, CA, U.S.A.) was used for analyzing the relative integrated density values. β-actin served as the control.
Enhanced chemiluminescence
Manufactured by CWBIO
Sourced in China
Enhanced chemiluminescence is a laboratory equipment that utilizes the principle of chemiluminescence to detect and measure the presence of specific molecules or proteins. It is a sensitive and versatile technique used in various applications, such as Western blotting, immunoassays, and reporter gene assays.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
2 protocols using enhanced chemiluminescence
1
Western Blot Analysis of Glioma Cells
2
Western Blot Analysis of Protein Signaling
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Cells were collected and lysed with ice-cold lysis buffer, and were then incubated on ice for 15 min. Protein levels were quantified by using bicinchoninic acid (BCA) assay. The amount of protein from each extract was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, USA). Membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST) for 30 min, and then incubated for 2 h at room temperature with antibodies at a suitable dilution as recommended (anti-p-TBK1, -TBK1, -p-IRF3, -IRF3, -Flag, -HA, and -Myc at 1:1000; anti-β-actin at 1:2000). The membranes were then incubated with the appropriate secondary antibody for 1 h at a dilution of 1:5000. After washing, signals were visualized using enhanced chemiluminescence (CWBIO, China) according to the manufacturer's protocols. Densitometry analysis of the indicate protein levels have been assessed by Image J software, and normalized to β-actin.
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