Mitochondria proteins were isolated from the right lung tissues of mice using a mitochondrial protein fraction kit (Beyotime, Shanghai, China) according to the manufacturer's protocols. Protein concentrations were determined by using a Pierce BCA Protein Assay Kit (Aidlab Biotechnologies, Beijing, China). Equal amounts of proteins were separated on a 12% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). After blocking with 5% fat-free milk for 1 h, the membrane was incubated with a rabbit anti-HO-1 antibody (1 : 2000, #10701, PTG, USA), mouse anti-MFN1 antibody (1 : 1000, #ab57602, Abcam, UK), rabbit anti-MFN2 antibody (1 : 1000, #12186, PTG, USA), rabbit anti-OPA1 antibody (1 : 1000, #ab157457, Abcam, UK), rabbit anti-DRP1 antibody (1 : 1000, #ab184247, Abcam, UK), rabbit anti-FIS1 antibody (1 : 1000, #ab71498, Abcam, UK), and mouse anti-β-actin antibody (1 : 8000, #KM9001, Tianjin Sungene Biotech, CN) at 4°C overnight. After being washed four times with TBST, the membrane was incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (1 : 3000, #E030130, Earthox, USA) at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence (#3300 Mini, Clinx Science Instruments, CN), and the relative densities of the bands were quantified by using the ImageJ software.
3300 mini
Manufactured by Clinx
Sourced in United States, China
The 3300 Mini is a compact and versatile lab equipment designed for accurate measurements. It features precise digital display and intuitive controls for efficient operation.
Automatically generated - may contain errors
2 protocols using 3300 mini
1
Isolation and Analysis of Mitochondrial Proteins
2
Protein Extraction and Western Blot Analysis
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Primary hepatocytes, Huh7 cells, the liver, and adipose tissues were extracted in RIPA lysis buffer containing protease and phosphatase inhibitors. After centrifugation at 12,000 × g for 15 min at 4 °C, the supernatants were assayed to determine the protein concentration using the BCA method. The protein was solubilized in SDS loading buffer and heated in a metal bath for 10 min. Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Missouri, USA, 0.45 µm pore size). After sealing with 5% skimmed milk powder, the membranes were washed and incubated with the corresponding primary antibodies overnight at 4 °C. After incubation with corresponding secondary antibodies (Biomike, China, 1:10,000) for 1 h at room temperature, the signals were developed using an ECL luminescent kit (ZENBIO, #17046, China) and detected using an enhanced chemiluminescence detection system (Clinx, 3300 Mini, China). Primary antibodies applied in the study are listed in Table 2. The protein bands were analyzed using Quantity one (4.6.2), and α-tubulin protein was used as an internal control.
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