Western blot analysis was performed as previously described (24 (link)). Briefly, SW480 cells were homogenized in phosphate-buffered saline (PBS) containing a protease inhibitor cocktail (Beyotime Institute of Biotechnology, Shanghai, China). The samples were incubated overnight at 4°C with rabbit anti-p53 antibody, -acetyl p53, -SIRT1, or -acetyl p21 antibody (all from Cell Signaling Technology Inc., Danvers, MA, USA). The antibody signal was detected using a Chemiluminescent Detection kit according to the manufacturer’s protocol (Beyotime Institute of Biotechnology, Jiangsu, China). The relative band intensities in the blots were determined using the Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA).
Chemiluminescent detection kit
Manufactured by Beyotime
Sourced in China
The Chemiluminescent Detection kit is a laboratory equipment used for detecting and quantifying various biomolecules, such as proteins, nucleic acids, and enzymes, through chemiluminescent signals. The kit provides the necessary reagents and protocols to enable sensitive and accurate detection of target analytes in various applications, including Western blotting, ELISA, and other immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Sirt1, by Cell Signaling Technology (1 mentions)
Acetyl p53, by Cell Signaling Technology (1 mentions)
Rabbit anti p53 antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Gel doc system, by Bio-Rad (1 mentions)
Bicinchoninic acid protein assay reagent, by Beyotime (1 mentions)
Nitrocellulose membrane, by Sangon (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Bca protein assay reagent kit, by Beyotime (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
4 protocols using chemiluminescent detection kit
1
Western Blot Analysis of p53 and Acetylation
2
Quantitative Protein Analysis Protocol
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Protein quantification was performed in the cell lysate supernatant by Micro Protein Assay Kit (Thermo Fisher Scientific, CA, USA). 10 μg of total cell protein sample was analyzed through 12% SDS-PAGE. After the transfer to PDVF membrane, the membrane was subjected to 1-h blocking using 5% BSA solution at ambient temperature. Proteins were detected with corresponding antibodies (1:1000 dilutions, all from Abcam) at 4 °C for 18 h. The membrane was further labeled with secondary antibodies conjugated with HRP to detect primary antibodies. Signal development was performed using a chemiluminescent detection kit (Beyotime, Beijing, China) and the images of bands were recorded on the BioRad gel doc system (BioRad, CA, USA).
3
Western Blot Analysis of Acetyl-p53, Bcl-2, and SIRT1
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PC12 cell lysates were extracted using a BCA Protein Assay Reagent kit (Beyotime Institute of Biotechnology, Shanghai, China), following mimic or inhibitor treatment. Protein concentration was determined using the bicinchoninic acid protein assay reagent (Beyotime Institute of Biotechnology). Equal quantities of protein from each sample were loaded and electrophoresed on 10% SDS-PAGE gels (Beyotime Institute of Biotechnology), and transferred to nitrocellulose membranes [Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China]. After blocking with non-fat dried milk, membranes were probed with polyclonal rabbit anti-human acetyl-p53 [dilution, 1:1,000 (cat. no. CS2525); Cell Signaling Technology, Inc., Danvers, MA, USA], polyclonal rabbit anti-human Bcl-2 [dilution, 1:1,000 (cat. no. BS4023); Bioworld Technology, Inc., St. Louis Park, MN, USA] or polyclonal rabbit anti-human SIRT1 [dilution, 1:1,000 (cat. no. CS2327); Cell Signaling Technology, Inc.] antibodies. Antibody signals were visualized using a Chemiluminescent Detection kit, according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Experiments with blank and negative controls were conducted in parallel. The relative band intensities of the blots were quantified with Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA, USA)..
4
Hippocampal Protein Expression Analysis
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The hippocampal tissue was triturated and homogenized in radio immunoprecipitation assay (RIPA) buffer, and the lysate was centrifuged at 12,000 ×g at 4 °C for 25 min. The protein concentration was determined using a BCA protein assay. Next, the samples were mixed with 5× loading buffer and heated at 99 °C for 10 min. After that, equal amounts of proteins (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with the primary antibodies anti-P2X4 and anti-NLRP3 (1:1000, Cell Signaling Technology, USA) at 4 °C overnight. Subsequently, the membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:5000, Proteintech, China) for 1 h. Finally, a chemiluminescent detection kit (Beyotime Biotechnology, Jiangsu, China) was utilized to observe the bands, and ImageJ software was employed for protein quantification.
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