The cells were lysed and homogenized using radioimmunoprecipitation assay buffer (Cell Signaling Technology), and the protein concentration was measured by an Enhanced Bicinchoninic acid Protein Assay Kit (Beyotime). Equal amounts of protein extracts were separated by electrophoresis on a 12% NuPAGE Bis-Tris Mini Gel (Invitrogen) and then transferred to nitrocellulose membranes (GE Healthcare) (24 (link)). The membranes were blocked for 1 hour in Tris-buffered saline containing 5 % bovine serum albumin and then probed with antibodies. Antibodies against SRM (Cat#: 19858), ODC1 (Cat#: 17003), SMOX (Cat#: 15052), iNOS (Cat#: 18985), IL-6 (Cat#: 21865), and GAPDH (Cat#: 60004) were purchased from Proteintech, and nuclear factor kappa B (NF-κB) p65 (Cat#: 8242S) was purchased from Cell Signaling Technology. The membranes were then probed with secondary antibodies conjugated to horseradish peroxidase. Next, the immunoblots were developed as recommended in the ECL Advance Western blot detection kit (Amersham Biosciences).
Ecl advance western blot detection kit
Manufactured by Cytiva
Sourced in United States
The ECL Advance Western blot detection kit is a lab equipment product designed for the detection and visualization of proteins in Western blot analysis. It provides a chemiluminescent-based detection method to enable sensitive and quantitative protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (3 mentions)
Mini protean tgx gel, by Bio-Rad (3 mentions)
Enhanced bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Interleukin 6 (il 6), by Proteintech (1 mentions)
Nupage bis tris mini gel, by Thermo Fisher Scientific (1 mentions)
Nuclear factor kappa b nf κb p65, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Bradford detection kit, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
5 protocols using ecl advance western blot detection kit
1
Protein Expression Analysis Using Western Blot
2
Western Blot Protein Quantification
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Each sample was lysed in TNEV buffer containing a cocktail of protease and phosphatase inhibitors. Protein concentration was determined with Bio-Rad Protein Assay kit. Then, the lysate was added at the same volume as the 2× SDS sample loading buffer with bromophenol blue. Equal amounts of total protein were separated by SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. The membranes were incubated with primary antibody for 1 h at 37°C, and then with HRP-conjugated secondary antibody for an additional 1 h at 37°C. The membranes were detected with ECL Advance Western Blot Detection Kit (Amersham, Marlborough, MA, USA).
3
Protein Expression Analysis in Muscle and Nerve Tissue
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Protein lysates were obtained by homogenization of mice tibialis anterior (TA) muscles and sciatic nerves in homogenization buffer as previously described 26 . Analysis of protein components (15 μg for tissue extracts) was performed by Mini‐PROTEAN® TGX™ Gels (BioRad, USA) and transfer onto nitrocellulose membranes (Amersham Biosciences, USA). After saturation with blocking agent, blots were incubated overnight at 4°C with the specified antibody against anti‐P2X7, rabbit (1:500 Alomone); anti‐Pax7, mouse (1:100 HDB); anti‐MyoG, mouse (1:10 HDB); anti‐GYS, rabbit and anti‐pGYS, rabbit (1:1000 Cell Signaling). Primary antibodies were then detected with HRP‐conjugated secondary antibodies and visualized using ECL Advance Western blot detection kit (Amersham Biosciences, USA). Signal intensity quantification was performed by Kodak Image Station analysis software.
4
Protein Extraction and Analysis
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Cells in serum-free medium were harvested in ice-cold RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) added with protease inhibitor cocktail (Sigma Aldrich). Lysates were kept for 30 min in ice and then centrifuged for 10 min at 14,000× g at 4 °C. Protein lysates were also obtained by mice lumbar spinal cords segments and cortex in homogenization buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 1% Triton X-100, 10 mM EDTA) added with protease inhibitor cocktail (Sigma Aldrich). After sonication, lysates were kept for 30 min in ice and then centrifuged for 20 min at 14000× g at 4 °C. Supernatants were collected and assayed for protein content by the Bradford detection kit (Bio-Rad Laboratories, Hercules, CA, USA.). Analysis of protein components was performed by Mini-PROTEAN® TGX™ Gels (BioRad) and transferred onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). After saturation with blocking agent, blots were probed overnight at 4 °C with the specified antibody, and then incubated for 1 h with HRP-conjugated secondary antibodies and visualized using ECL Advance Western blot detection kit (Amersham Biosciences). Signal intensity quantification was performed by Kodak Image Station analysis software. Values were normalized with mouse anti-GAPDH (1:2500, Calbiochem).
5
Western Blot Analysis of Microglia Proteins
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Microglia in serum-free medium were harvested in SDS Laemmli sample buffer. Protein lysates were obtained by homogenization of mice lumbar spinal cords segments in homogenization buffer as described (Apolloni et al., 2014 (link)). Analysis of protein components (10 μg for tissue extracts) was performed by Mini-PROTEAN® TGX™ Gels (BioRad, USA) and transfer onto nitrocellulose membranes (Amersham Biosciences, USA). After saturation with blocking agent, blots were incubated overnight at 4°C with the specified antibody, then for 1 h with HRP-conjugated secondary antibodies and visualized using ECL Advance Western blot detection kit (Amersham Biosciences, USA). Signal intensity quantification was performed by Kodak Image Station analysis software.
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