Western blot was conducted as previously described [23 (link)]. Briefly, protein concentration in the supernatant of cells was determined using a BCA Protein Assay Kit (Servicebio). Proteins (30 μg) were separated on an SDS-PAGE gel (Beyotime) and transferred to PVDF membranes. Subsequently, the membranes were incubated overnight with primary antibodies against anti-Bcl-2, anti-Bax, anti-caspase 3, anti-caspase 9, anti-Cytochrome c (Cyt-c), anti-apoptosis-inducing factor (AIF), anti-Smac/DIABLO, anti-Tnmd, anti-MMP-1, anti-Col I, anti-Col III, anti-Dynamin-related protein 1 (Drp1), anti-Mitofusin 2 (Mfn2), and anti-β-actin. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 22–26 °C. Protein bands were visualized using the BeyoECL Moon chemiluminescence system (Beyotime). All antibody information and dilutions are listed in Additional file 5 : Table S2.
Beyoecl moon chemiluminescence system
Manufactured by Beyotime
The BeyoECL Moon chemiluminescence system is a lab equipment product designed for the detection and analysis of chemiluminescent signals. It utilizes a sensitive camera and advanced optics to capture and quantify chemiluminescent reactions, which are commonly used in various biochemical and molecular biology applications.
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Lab products found in correlation
Sds page gel, by Beyotime (1 mentions)
Bca protein assay kit, by Wuhan Servicebio Technology (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Bca assay kit, by Wuhan Servicebio Technology (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Gb23301, by Wuhan Servicebio Technology (1 mentions)
Ab124773, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab256470, by Abcam (1 mentions)
Anti bax, by Wuhan Servicebio Technology (1 mentions)
Gb15001, by Wuhan Servicebio Technology (1 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (1 mentions)
2 protocols using beyoecl moon chemiluminescence system
1
Comprehensive Western Blot Analysis
2
Western Blot Analysis of Apoptosis and Mitochondrial Dynamics
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After extracting the proteins with RIPA buffer and protease inhibitor, the total protein contents were measured using a BCA assay kit (Servicebio). By normalizing protein content, all samples had the same quality and volume for further analysis. A wet-transfer method was used to separate proteins by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transfer them to polyvinylidene fluoride membranes. The membranes were blocked in pure methanol before use and then blocked in 1 × TBS containing 3% bovine serum albumin (Solarbio, Beijing, China). The membranes were then incubated overnight with primary antibodies: Anti-Bax (GB11690; Servicebio), anti-Bcl-2 (ab196495; Abcam), anti-caspase-3 (GB11767C; Servicebio), anti-superoxide dismutase 2 (SOD2) (GB111875; Servicebio), anti-glutathione peroxidase 3 (GPx-3) (ab256470; Abcam), anti-dynamin-related protein 1 (DRP1) (8570S; CST), anti-mitofusin 2 (MFN2) (ab124773; Abcam), and anti-β-Actin (GB15001; Servicebio) antibodies. After washing with TBS-T, they were incubated with appropriately diluted horseradish peroxidase (HRP) conjugated Goat Anti-Rabbit immunoglobulin (Ig)G (GB23303; Servicebio) or HRP conjugated Goat Anti-Mouse IgG (GB23301; Servicebio) as the secondary antibody. Protein bands were visualized using the BeyoECL Moon chemiluminescence system (Beyotime).
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