Total protein was extracted from cell lines (SKOV3 and HO8910) and mouse tissue samples (250 mg/sample) using radio immunoprecipitation assay lysis buffer (cat. no. P0013D; Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Complete™ Mini; Roche Applied Science) at 4 °C for 10 min. Protein concentration was measured using a bicinchoninic acid protein assay kit (cat. no. P0012S; Beyotime Institute of Biotechnology). Proteins (40 µg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Beyotime Institute of Biotechnology). Membranes were blocked with tris-buffered saline containing 5% non-fat milk (weight/volume) for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit (cat. no. ab6721 and ab6728; 1:2,000; Abcam) secondary antibodies for 1 h at room temperature. The bands were visualised through enhanced chemiluminescence detection (Beyotime Institute of Biotechnology) with a ChemiDoc™ MP Imaging System and analysed using the Image Lab software (version 3.0; Bio-Rad Laboratories, Inc.).
Enhanced chemiluminescence detection
Manufactured by Beyotime
Sourced in China
Enhanced chemiluminescence detection is a laboratory equipment used to analyze and quantify the presence of specific proteins or molecules in a sample. It utilizes the principle of chemiluminescence, where a chemical reaction emits light that can be detected and measured. This equipment provides a sensitive and accurate method for protein detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Ab6721, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Complete mini, by Roche (1 mentions)
Ab6728, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Beyotime (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
2 protocols using enhanced chemiluminescence detection
1
Western Blot Analysis of Cellular Proteins
2
CagA Protein Expression Verification
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Protein expression of the CagA transgene in AGS cells was verified by western blotting. In brief, total proteins were extracted from nontransfected AGS cells, vector control-transfected AGS cells, and AGS cells transfected with CagA-encoding plasmid. The CagA levels were assessed by electrophoresis on a polyacrylamide gel, followed by transfer to a polyvinylidene difluoride (PVDF) membrane (Invitrogen). After washing three times with Tris-buffered saline containing Tween 20 for 15 min, the PVDF membrane was incubated with the secondary antibody (sc-25766; Invitrogen) for 1 h at room temperature, followed by repeated washing and enhanced chemiluminescence detection (Beyotime Biotechnology, Shanghai, China), according to the manufacturer's specifications and routine procedures using a gel imaging analyzer (Chemidoc XRS+, Bio-Rad, Hercules, CA, USA).
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