The proteins of mitochondria are important components of the apoptotic pathway. C26 cells (1×105 cells per well in 6-well plates) were transfected with the mBim/DMP-039 complex. After 48 hours, cells were collected, and protein samples were extracted with RIPA/IP (Beyotime). In order to ensure that the loading volume for each protein sample was consistent, it was necessary to determine the concentration of total proteins with Coomassie brilliant blue G-250 (Beyotime). Next, the proteins were separated by polyacrylamide gel electrophoresis (Beyotime) and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The PVDF membrane was blocked with 5% non-fat milk (Servicebio Technology, Wuhan). Then we probed the target protein with the primary antibody overnight at 4°C as follows: Bim (1:1000, Abcam), Bcl-2 (1:2000, Abcam), cytochrome c (1:5000, ab133504), caspase-9/cleaved caspase-9 (1:2000, Abcam), and β-actin (1:1000, Abcam). The PVDF membrane was washed and incubated with goat-rabbit (1:10,000, Cell Signaling Technology) secondary antibody. The target proteins were detected using a chemiluminescence system (ChemiScope Touch, CLINX, Shanghai).
Polyacrylamide gel electrophoresis
Manufactured by Beyotime
Sourced in China
Polyacrylamide gel electrophoresis is a laboratory technique used to separate and analyze biomolecules, such as proteins or nucleic acids, based on their size and charge. It utilizes a polyacrylamide gel matrix and an electric current to facilitate the migration of the biomolecules through the gel.
Automatically generated - may contain errors
Lab products found in correlation
Cytochrome c, by Abcam (1 mentions)
Chemiscope touch, by Clinx (1 mentions)
Coomassie brilliant blue g 250, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Goat anti rabbit immunoglobulin g, by Santa Cruz Biotechnology (1 mentions)
Bca protein quantitation kit, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Cold cell lysis buffer, by Beyotime (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico substrate, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Keygen Biotech (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Immobilon, by Merck Group (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
5 protocols using polyacrylamide gel electrophoresis
1
Mitochondrial Proteins in Apoptosis
2
Western Blot Analysis of Protein Expressions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples (either BV2 cells or rat retinas) were collected to a glass homogenizer and grinded with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor (Beyotime), and then incubated on ice for 15 min. The supernatant was isolated by supercentrifuge at 15,000 g/min for 5 min at 4°C. The total protein concentration in each sample was quantified using the bicinchoninic acid (BCA) Protein Quantitation Kit (Beyotime). The protein samples were added with 5× SDS (Sodium Dodecyl Sulfate) loading buffer (Beyotime) in a 4:1 ratio and then subjected to SDS-PAGE (Polyacrylamide Gel Electrophoresis; Beyotime). After electroblotting transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad), the 5% non-fat milk in tris-buffered saline Tween (TBST) was used to block and different primary antibodies were incubated with at 4°C overnight (Table 1 ). Washed the membranes with TBST three times and incubated with horseradish peroxidase-conjugated sheep anti-mouse (1:3000; Santa Cruz Biotechnology) or goat anti-rabbit immunoglobulin-G (1:3000; Santa Cruz Biotechnology) as secondary antibodies for 2 h at 25°C. After washing three times with TBST, chemiluminescence detection reagents were used to visualize the bands on the membranes. β-actin was used as a internal control.
3
Western Blot Analysis of Osteoclast Proteins
4
Western Blotting Analysis of Acetylated H3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 50 µg of whole cell lysate was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology, Haimen, China) at 60 V for 30 min and 110 V for 1 h, and transferred onto a polyvinylidene difluoride membrane (Immobilon; EMD Millipore, Billerica, MA, USA). After blocking in 5% non-fat dry milk, the membranes were incubated with primary antibodies overnight at 4°C, as follows: Rabbit polyclonal anti-acetylated H3 (Ac-H3, 1:10,000, EMD Millipore; cat. no. 382158) and rat monoclonal anti-laminin β1 (1:500, Abcam, Cambridge, MA, USA; cat. no. ab44941) overnight at 4°C. Polyclonal horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; OriGene Technologies, Beijing, China; cat. no. TA130023) was used as the secondary antibody, and the bands were detected using an Enhanced Chemiluminescence Detection kit (Nanjing KeyGen Biotech Co., Ltd. Nanjing, China) and analyzed by Quantity One Software (version 4.62; Bio-Rad Laboratories, Inc.). Protein expression was determined relative to expression of laminin β1.
5
Exosomal Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from exosomes performed using radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO). The protein samples were separated by 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (Beyotime, Shanghai, China) and subsequently transferred onto PVDF membranes (Life Technology, Waltham, MA). After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with the appropriate primary antibodies, CD63 (1:1000 dilution, CST, #52090), CD9 (1:500 dilution, CST, #98327), and Alix (1:1000 dilution, CST, #92880). Following the primary antibody incubation, the membranes were incubated with the corresponding secondary antibody for an additional 1 hour at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!