Fibroblasts (5x106) were cultured in 60-mm dishes and lysed using RIPA lysis buffer with protease and phosphatase inhibitors (ComWin Biotech). Protein lysates were centrifuged at 14,000 x g/min at 4˚C for 30 min, and a Bradford Protein Assay kit (ComWin Biotech) was used to determine the concentration of proteins. A total of 20 µg protein was separated using 12% SDS-PAGE and transferred to PVDF membranes using a Bio-Rad transfer system (Bio-Rad Laboratories, Inc.). Subsequently, the PVDF membranes were blocked in 5% fat-free milk for 2 h at room temperature and then incubated with primary antibodies at 4˚C overnight. After incubation with the HRP-linked secondary antibody (1:2,000; cat. no. ab97051l; Abcam) at room temperature for 1 h, the membranes were washed with 0.5% TBST and the BeyoECL Plus solution (Beyotime Institute of Biotechnology) was used to visualize the bands. For signal detection, Quantity One software (version 4.6.7; Bio-Rad Laboratories, Inc.) was used for semi quantitative analysis. The primary antibodies used are as follows: TNF-α (1:10,000; cat. no. ab109322; Abcam), IL-1β (1:1,000; cat. no. ab234437; Abcam), Nlrp3 (1:1,000; cat. no. ab263899; Abcam), IκBα (1:1,000; cat. no. ab32518; Abcam) and anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc.).
Beyoecl plus solution
Manufactured by Beyotime
Sourced in China
BeyoECL Plus solution is a chemiluminescent substrate solution designed for the detection of horseradish peroxidase (HRP) in Western blotting applications. The solution produces a luminescent signal when the HRP enzyme catalyzes the oxidation of the substrate, allowing for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by CoWin Biotech (1 mentions)
Transfer system, by Bio-Rad (1 mentions)
Il 1β, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Anti β actin, by Abcam (1 mentions)
Nlrp3, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
3 protocols using beyoecl plus solution
1
Evaluating Inflammatory Protein Expression
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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After AF cells were cultured in the conditioned media for 3 days, total protein
was extracted using RIPA lysis buffer (Beyotime, China). Protein concentration
was measured using an Enhanced BCA Protein Assay Kit (Beyotime, China). Then,
equal protein samples in each group were separated by sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred onto
polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, U.S.A.). And
thereafter, the PVDF membranes were incubated with primary antibodies overnight
at 4°C, followed by incubation with horseradish peroxidase-labeled
secondary antibodies for 2 h at room temperature. Immunoreactive protein bands
on the PVDF membrane were developed by BeyoECL Plus solution (Beyotime, China).
Finally, densitometry analysis of protein bands was performed using the Image J
software.
was extracted using RIPA lysis buffer (Beyotime, China). Protein concentration
was measured using an Enhanced BCA Protein Assay Kit (Beyotime, China). Then,
equal protein samples in each group were separated by sodium dodecyl
sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred onto
polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, U.S.A.). And
thereafter, the PVDF membranes were incubated with primary antibodies overnight
at 4°C, followed by incubation with horseradish peroxidase-labeled
secondary antibodies for 2 h at room temperature. Immunoreactive protein bands
on the PVDF membrane were developed by BeyoECL Plus solution (Beyotime, China).
Finally, densitometry analysis of protein bands was performed using the Image J
software.
3
Exosomal Protein Characterization by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins were extracted from the exosomes and cells using radio immunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich). The protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime). Equal amounts of protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked using 5% nonfat milk and incubated overnight at 4°C with the anti-CD9 and anti-CD63 primary antibodies. The following day, the membranes were incubated for 2 h with horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG at 25°C room temperature. BeyoECL Plus solution (Beyotime) was used for visualizing the protein bands.
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