Radio immunoprecipitation assay lysis buffer (Yeasen, China) was applied to extract proteins from PC-12 cells or spinal cord tissues. Protein concentration was determined with a bicinchoninic acid kit (Yeasen, China). Equal amounts of protein (30 μg) in all samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride films (Yeasen, China). Nonspecific protein binding was blocked using 5% nonfat milk in 0.1% Tris Buffered Saline Tween for 2 h at room temperature. Next, the membranes were incubated with primary antibodies against interleukin (IL)-6 (1:1000, Abcam), IL-1β (1:1000, Abcam), tumor necrosis factor alpha (TNF-α; 1:1000, Abcam), B-cell lymphoma-2 (Bcl-2; 1:1000, CST), Bcl-2 associated X (Bax; 1:1000, CST), pro caspase-3 (1:1000, Abcam), cleaved caspase-3 (1:500, Abcam), NOX4 (1:2000, Abcam) and GAPDH (1:1000, CST) overnight at 4°C. Next, the horseradish peroxidase-marked secondary antibody was added and incubated for 1 h. The bands were visualized with the enhanced chemiluminescent kit (Yeasen, China). The expression levels of proteins were analyzed by the Image J software (version ImageJ 1.44P; National Institute of Health) to determine gray density.
Enhanced chemiluminescent kit
Manufactured by Yeasen
Sourced in China
The Enhanced chemiluminescent kit is a laboratory equipment designed for the detection and quantification of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to generate a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 6 (il 6), by Abcam (1 mentions)
Cleaved caspase 3, by Abcam (1 mentions)
Bicinchoninic acid kit, by Yeasen (1 mentions)
Il 1β, by Abcam (1 mentions)
Tnf α, by Abcam (1 mentions)
Pro caspase 3, by Abcam (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Yeasen (1 mentions)
Enhanced bca protein assay reagent, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Kgp113k, by Keygen Biotech (1 mentions)
2 protocols using enhanced chemiluminescent kit
1
Western Blot Analysis of Inflammatory and Apoptotic Markers
2
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Tissues and cells were lysed with RIPA lysis buffer (Beyotime) supplemented with protease inhibitors and phosphatases inhibitors. Total protein concentration was detected using Enhanced BCA Protein Assay Reagent (Beyotime). Denatured proteins (25 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transfected to PVDF membranes (Keygen Biotech, KGP113K). After blocking with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4°C with the indicated primary antibodies, followed by incubation with a goat anti-rabbit/mouse antibody for 1 h at room temperature. Finally, the protein bands were visualized using the enhanced chemiluminescent kit (Yeasen), and the levels of proteins were normalized to the GAPDH expression.
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