Western blot analysis was performed as previously described[10 (link)]. In brief, immunoprecipitated complexes were separated by 10% SDS–PAGE and aliquots of cell lysates were separated using SDS-PAGE on a 12% polyacrylamide gel, then transferred to a PVDF membrane. Blots were blocked with approximately 5% nonfat dry milk for 2 h, shaken at room temperature, and then incubated with primary anti-S100A9, anti-Vimentin, anti-Annexin A1 or anti-β-actin antibody at 4°C overnight followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 2 h and shaking at room temperature. Proteins were detected using an enhanced chemiluminescence Western blot system (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the band intensity was measured using densitometry combined with Image- Pro Plus 6.0 software (National Institutes of Health, Bethesda, MD, USA).
Enhanced chemiluminescence western blot system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced Chemiluminescence Western Blot System is a laboratory equipment designed for the detection and visualization of proteins in Western blot analysis. It utilizes a chemiluminescent substrate to produce a light signal that is proportional to the amount of target protein present, allowing for sensitive and quantitative protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Ab39012, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab41037, by Abcam (1 mentions)
Ab194583, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
3 protocols using enhanced chemiluminescence western blot system
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Targets
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Cells were harvested and lysed in modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.25% sodium deoxycholate, 1 mM DDT, 10 mM NaF, 1 mM PSMF, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mM Na3VO4). Following lysis, the lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 12% polyacrylamide gel, and the proteins were transferred to a polyvinylidene difluoride nylon membrane and probed with primary antibodies for target proteins overnight at 4 °C. The target proteins were after incubation with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The signals were revealed using an Enhanced Chemiluminescence Western Blot System (Thermo Scientific, Rockford, IL, USA).
3
Western Blot Analysis of Autophagy and Apoptosis Markers
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Proteins from cell lysates were extracted by 7.5 to 12.5% SDS–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (0.45 μm; Millipore, Bedford, MA, USA). The membranes were blocked by applying 5% nonfat dry milk in tris-buffered saline with Tween-20 for 0.5 hours. Then, they were incubated at 4°C overnight with anti-PTX3 antibodies (1:1000; 13797-1-AP, Proteintech), anti-ATG5 antibodies (1:1000; 12994, Cell Signaling Technology), anti-ATG7 antibodies (1:2000; abs135650, Absin), anti–Beclin-1 antibodies (1:1000; 3738, Cell Signaling Technology), anti-LC3B antibodies (1:1000; 3868, Cell Signaling Technology), anti-Bcl2 antibodies (1:500; ab194583, Abcam), anti-BAX antibodies (1:500; ab32503, Abcam), anti–caspase-3 antibodies (1:500; ab13847, Abcam), anti-COL2 antibodies (1:500, ab34712, Abcam), anti-ACAN antibodies (1:1000; ab36861, Abcam), anti-MMP13 antibodies (1:1000; ab39012, Abcam), and anti-ADAMTs antibodies (1:1000; ab41037, Abcam), followed by incubation with the corresponding horseradish peroxidase–conjugated secondary antibody (1:4000; Santa Cruz Biotechnology) for 1 hour. Antibody reactivity was visualized using the Enhanced Chemiluminescence Western Blot System (Thermo Fisher Scientific, Pierce, Rockford, IL, USA).
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