Radioimmunoprecipitation assay (RIPA) buffer mixed with protease and phosphatase inhibitor mixtures was used to extract the proteins, and the proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, USA) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% to 12.5% polyacrylamide gels). Primary antibodies were anti-COL2α1 antibody (1:1,000, Abcam Cat# ab34712, RRID: AB_731688), anti-MMP-13 antibody (1:1,000, Abcam Cat# ab39012, RRID: AB_776416), anti-a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) antibody (1:250, Abcam Cat# ab41037, RRID: AB_2222327), anti-β-actin antibody (1:1,000, Abcam Cat# ab8226, RRID: AB_306371), and anti-KLF5 antibody (1:2,000, Abcam Cat# ab137676, RRID: AB_2744553; all Abcam). Secondary antibody was rabbit anti-mouse IgG H&L antibody (1:5,000, Abcam Cat# ab6728, RRID: AB_955440; Abcam). Primary and secondary antibodies were used to detect the corresponding proteins. The results were obtained using the Enhanced Chemiluminescence (ECL) Western blot System (Amersham Biosciences, China).
Ecl western blot system
Manufactured by Cytiva
Sourced in China
The ECL Western Blot System is a laboratory equipment used for the detection and analysis of proteins separated by gel electrophoresis and transferred to a membrane. It utilizes a chemiluminescent detection method to visualize the target proteins. The system provides the necessary components for the Western blotting process, including the reagents and the required equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ab34712, by Abcam (1 mentions)
Ab39012, by Abcam (1 mentions)
Ab137676, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab6728, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Runx2, by Santa Cruz Biotechnology (1 mentions)
Osterix, by Santa Cruz Biotechnology (1 mentions)
3 protocols using ecl western blot system
1
Western Blot Analysis of Cartilage Regulatory Proteins
2
Protein Analysis Using SDS-PAGE and Western Blot
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Protein analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Protein samples were subjected to 10% or 12% SDS-PAGE and were stained with Coomassie Brilliant Blue R-250 [33 (link)]. Western blot analysis was carried out using the enhanced chemiluminescence (ECL) Western blot system (Amersham Biosciences) with anti-BivCaE antibodies. Glycoprotein was stained using Gel Code Glycoprotein Staining Kit (Pierce, Rockford, IL, USA).
3
Protein Extraction and Western Blot Analysis
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The protein samples were extracted from osteoblasts via procedures that have previously been described in detail (Yang et al. 2007) (link). Protein samples (approximately 50 mg) were fractionated by SDS-PAGE (7.5-10% polyacrylamide gels). Separated proteins were blot transferred onto a nitrocellulose membrane. After blocking with 0.1% Tween 20 and 5% nonfat dry milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated overnight at 4 8C in one of the following primary antibodies as an internal control: FZD4 (1:400; Peprotech, Rocky Hill, NJ, USA), WNT2 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-catenin (1:200; Santa Cruz Biotechnology), Runx2 (1:200; Santa Cruz Biotechnology), Osterix (1:400; Santa Cruz Biotechnology), and b-actin (1:1000; Santa Cruz Biotechnology). The membrane was incubated with HRP-conjugated secondary antibody (1:2000) for 1 h and detected using the ECL Western Blot System (Amersham Biosciences).
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