The procedures of Western blot were adopted from a previous study [33 (link)]. RIPA lysate was utilized for extracting total proteins from cells. Cells suspended in RIPA buffer were lyzed on ice for 10 mins and lysed cells were centrifuged at 14,000 rpm for 10 mins. The supernatant containing total protein lysate was quantified by a BCA Protein assay kit (Beyotime Biotechnology P0009; Shanghai, China). Aliquots of proteins (30 µg) were seperated through 10% SDS-PAGE, followed by the transfer of proteins onto PVDF membranes. After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies overnight at 4°C.: B-cell lymphoma-2 (Bcl-2) (1:500; ab196495, Abcam, Cambridge, UK), BCL2-associated x protein (Bax) (1:1000; ab32053, Abcam), Cleaved caspase 3 (1:1000; ab231289, Abcam), MAPK1 (1:800; ab265600, Abcam) and β-actin (1:5000; ab179467, Abcam). The membrane was washed 3 times with TBST for 5 minutes each. After wash, the membrane was further incubated with goat anti-rabbit IgG H&L (Abcam; ab96899, 1:5000) for additional 1 h under ambient temperature. Enhanced chemiluminescence ECL reagent (Pierce, IL, USA) was employed to visualize protein bands. The protein bands image was captured using Gel Doc 100 system and the Quantity One® imaging software (Bio-Rad, CA, USA).
Ab231289
Manufactured by Abcam
Sourced in United Kingdom
Ab231289 is a lab equipment product offered by Abcam. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Ab196495, by Abcam (1 mentions)
Ab96899, by Abcam (1 mentions)
Ab265600, by Abcam (1 mentions)
Ab32053, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
Quantity one imaging software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ab199956, by Abcam (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
Ab68183, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab226983, by Abcam (1 mentions)
4 protocols using ab231289
1
Western Blot Analysis of Apoptosis Markers
2
Protein Expression Analysis in bEnd.3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
bEnd.3 cells were disrupted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime) plus 1% protease inhibitor (Invitrogen). Protein concentration was assessed using BCA protein assay kit (Invitrogen). 20 μg protein samples were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then electro-transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA), which was blocked with 5% skim milk for 1 h. After that, the membrane was incubated with primary antibodies, including anti-B cell leukemia/lymphoma 2 (anti-Bcl-2; ab59348; Abcam, Cambridge, MA, USA), anti-Bcl-2 associated X, apoptosis regulator (anti-Bax; ab182733; Abcam), anti-Cleaved caspase 3 (anti-Cleaved-cas3; ab231289; Abcam), anti-endothelial nitric oxide synthase (anti-eNOs, ab199956; Abcam), anti-EPHA4 (E6900; Sigma, St. Louis, MO, USA) and anti-GAPDH (ab8245; Abcam). After washing three times, horse radish peroxidase (HRP)-labeled secondary antibody (Abcam) was incubated with the membrane for 2 h. Protein signal was measured by the enhanced chemiluminescent visualization (ECL) system (Pierce, Rockford, IL, USA).
3
Western Blot Analysis of Neuronal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were obtained from lysates of primary rat cortical neurons and mouse brain tissue samples using RIPA lysis buffer (Beyotechnology, Shanghai, China). To measure protein expression levels, these samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were then transferred onto polyvinylidene difluoride membranes (Millipore, USA). After blocking with 5% non-fat milk at 4°C for 1 h, the membranes were incubated with anti-cleaved caspase-3 (1:1000, ab231289, Abcam, UK), anti-COX-2 (1:1000, ab179800, Abcam, UK), anti-SOX8 (1:1000, ab226983, Abcam, UK), antiβ-catenin (1:1000, ab68183, Abcam, UK), anti-lamin B1 (1: 1000, ab16048, Abcam, UK), and GAPDH (1:1000, ab8245, Abcam, UK) at 4°C overnight, followed by incubation with the appropriate secondary antibody at room temperature for 1 h. Glyceraldehyde 3-phosphate dehydrogenase was used as the endogenous control. Protein blots were visualized using an ECL chemiluminescence kit (Beyotechnology, Shanghai, China).
4
Exosome Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysis buffer (Lab-bio, China) added with the PMSF (Yuanye, China) were injected into the OC cells and exosomes. After ice bath for 30 min, the lysates were centrifuged at 12000×g and 4℃ for 8 min. The concentration and quality of the extracted protein was determined by spectrophotometry method. Then 15 μg of the protein was added to the loading well of 12% prefabricated gel and the electrophoresis was conducted. After that, the separated proteins in the gel were transferred to the nitrocellulose (NC) membrane (Millipore, USA). The NC membrane was soaked in the blocking buffer (1% BSA diluted by TBST) for 1 h. Then the NC membrane was immersed in different primary antibodies at 4℃ for overnight. After rinsing with TBST, the NC membrane was immersed in the secondary antibody solution for another 1 h. In the end, ECL solution (Invitrogen, USA) was dropped onto the membrane, and then development was performed with Bio-Rad gel imaging system (USA). Antibodies used in this research were all purchased from Abcam (UK), including anti-TSG101 (ab228013, 1:2000), anti-CD63 (ab321975, 1:1000), anti-GRP94 (ab3674, 1:3000), anti-Bax (ab263897, 1:2500), anti-Bcl2 (ab194583, 1:1500), anti-Cleaved Caspase-3 (ab231289, 1:800), anti-Cleaved Caspase-9 (ab2324, 1:800), anti-MMP2 (ab181286, 1:1000), anti-MMP9 (ab283575, 1:1500), goat anti-rabbit IgG (ab97051, 1:40000).
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!