Western blotting was performed using extracts from samples of the entire adult cochlea. A whole brain was removed from one animal. Tissues obtained from the two different regions were homogenized separately in ice-cold RIPA Lysis Buffer (50 mM Tri-HCl, pHs 7.6, 150 mM NaCl, 0.1% SDS, 1% NP40, and 0.1 mM EGTA). Homogenates were fractionated by SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. Membranes were blocked with 5% w/v nonfat dry milk and immunoblotting was performed using rabbit anti-IP3 polyclonal antibodies (1:1000; Abcam, Cambridge, MA, USA) and anti-β-Actin monoclonal antibody (1:1000; Biyuntian, Hangzhou, China) served as an internal loading control. The protein bands were visualized by the application of horseradish peroxidase-conjugated secondary antibody, and were detected using a chemiluminescent reagent (Biyuntian). Reaction product levels were quantified by the Quantity One System (Bio-Rad, Hercules, CA, USA).
Anti β actin monoclonal antibody
Manufactured by Beyotime
Sourced in United States
The Anti-β-Actin monoclonal antibody is a laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. It is a highly specific antibody that binds to the β-actin isoform, a ubiquitous cytoskeletal protein found in eukaryotic cells. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to analyze the expression levels of β-actin in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one system, by Bio-Rad (2 mentions)
Enhanced chemiluminescence reagent, by Vazyme (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Bhk 21, by National Collection of Authenticated Cell Cultures (1 mentions)
Ma 104, by National Collection of Authenticated Cell Cultures (1 mentions)
4 protocols using anti β actin monoclonal antibody
1
Analyzing IP3 Expression in Cochlear and Brain Tissues
2
Quantifying Cochlear Cx43 Expression
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Western blotting was performed using extracts from samples of the entire cochlea at P1. A whole brain was removed from one animal. Tissues obtained from the two different regions were homogenized separately in ice-cold RIPA Lysis Buffer (50 mM Tri-HCl, pH 7.6, 150 mM NaCl, 1% SDS, 1% Triton X-100, and 0.1 mM EGTA). Homogenates were fractionated by SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. Membranes were blocked with non-fat dried milk and immunoblotting was performed using rabbit anti-Cx43 antibodies (1:400; Invitrogen, Carlsbad, CA, USA) and anti-β-Actin monoclonal antibody (1:1000; Biyuntian, Hangzhou, China) served as an internal loading control. The protein bands were visualized by the application of horseradish peroxidase-conjugated secondary antibody, and were detected using a chemiluminescence reagent (Biyuntian). Reaction product levels were quantified by the Quantity One System (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Apoptosis Markers
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Cells were treated as indicated and then washed with PBS, lysed in Tris-lysis buffer (Beyotime, Shanghai, China) in an ice bath for 20 min, and centrifuged (12,000 × g, 10 min). The supernatant was mixed with 6 × SDS loading buffer, then boiled for 10 min, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat milk at 4°C overnight and incubated with different primary antibodies [rabbit anti-caspase-3, -8, and -9 polyclonal antibody (Abcam, United States) or anti-β-actin monoclonal antibody (Beyotime, Shanghai, China), all antibodies were diluted 1:1000] at 37°C for 2 h. The membranes were then washed with PBS containing 0.05% Tween-20 and reacted with corresponding horseradish peroxidase-conjugated secondary antibodies at 37°C for 1 h. All blots were developed using enhanced chemiluminescence reagents (Vazyme, Nanjing, China).
4
Porcine Rotavirus and Vesicular Stomatitis Virus Propagation
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MA104 and BHK-21 cells (obtained from China Center For Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) at 37 °C under 5% CO2. PRV (strain DN30209, isolated in a pig farm, in Heilongjiang province of China) [26 (link)] and vesicular stomatitis virus (VSV, strain Indiana, obtained from prof. Joerg Glende, University of Veterinary Medicine Hannover, Germany) were propagated on the MA104 cells and BHK-21 cells, respectively, as previously described [26 (link), 27 (link)]. In as much as previous reports showed that VSV infection on BHK-21 is not affected by depletion of cholesterol [27 (link)], we used VSV as a negative control for our studies about relationship between lipid rafts and virus infection.
Rabbit polyclonal antibodies to VP4 and VP7 were generated in our laboratory using previously described methods [26 (link), 28 ]. Anti-VSV G-protein polyclonal antibody was purchased from Abcam. An anti-β-actin monoclonal antibody was purchased from Beyotime. The secondary antibodies were purchased from BD Biosciences. Both MβCD and cholesterol were purchased from Sigma and reconstituted in DMEM and alcohol, respectively.
Rabbit polyclonal antibodies to VP4 and VP7 were generated in our laboratory using previously described methods [26 (link), 28 ]. Anti-VSV G-protein polyclonal antibody was purchased from Abcam. An anti-β-actin monoclonal antibody was purchased from Beyotime. The secondary antibodies were purchased from BD Biosciences. Both MβCD and cholesterol were purchased from Sigma and reconstituted in DMEM and alcohol, respectively.
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