Proteins were extracted from preadipocytes and adipocytes. After detecting the total protein concentration, the protein was denatured at 95°C for 5 min with a protein loading of 50 μg. Subsequently, SDS-PAGE electrophoresis was performed with 10% of the isolate gel and 4% of the concentrate gel, and electrophoresis at 40 V for 25 min in the concentrate gel and 100 V for 80 min in the isolate gel. Then, the protein was transferred to the PVDF membrane and immersed in the closure solution at 37°C for 1.5 h. Then, it was incubated in monoclonal rabbit anti-ENTPD1, anti-USP2, and anti-PGAM2 (1:1000; Abcam, Cambridge, United Kingdom) and monoclonal mouse anti-β-actin (1:5,000; Beyotime, Shanghai, China). Finally, the membranes were incubated for 1.5 h at 37°C by adding an HRP-labeled goat secondary antibody and images captured using a Chemi Doc System (Bio-Rad, Hercules, CA). Grayscale values of proteins were evaluated by ImageJ (https://imagej.nih.gov/ij/ ).
Monoclonal mouse anti β actin
Manufactured by Beyotime
Sourced in United States
Monoclonal mouse anti-β-actin is a laboratory reagent used to detect and quantify the presence of the beta-actin protein in biological samples. It is a highly specific antibody that binds to the beta-actin protein, allowing for its identification and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc system, by Bio-Rad (1 mentions)
Rabbit monoclonal anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Chemiluminescence detection reagent, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by Beyotime (1 mentions)
Proteinase inhibitors, by Sangon (1 mentions)
Hrp conjugated goat anti mouse igg h l, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
5 protocols using monoclonal mouse anti β actin
1
Protein Expression Analysis in Adipogenesis
2
Western Blot Assay Protocol for Protein Analysis
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Western blot assay was performed as previously described (12 (link)). In brief, the total protein was extracted and separated by SDS-PAGE. Then the separated proteins were transferred to PVDF membranes. The membranes were incubated with the corresponding primary antibodies followed by the relevant secondary antibody. Primary antibodies used in the present study were monoclonal rabbit anti-E-cadherin (1:1,000), anti-vimentin (1:500) (both from Cell Signalling Technology, Danvers, MA, USA) and monoclonal mouse anti-β-actin (1:1,000; Beyotime).
3
Osteogenic Differentiation of hPDLSCs
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The hPDLSCs were lysed for 30 min at 7 days post osteogenic induction. Cell extracts containing 40 µg total protein were loaded and separated by 4–20% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SurePAGE, Ubiotechnology, Guangdong, China) and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline +0.1% Tween-20 (TBST) containing 5% bovine serum albumin (BioFroxx, Einhausen, Germany) for 2 h at 20 °C–25 °C after transfer. The membranes were immunoblotted with the following primary antibodies at a 1:1,000 dilution: polyclonal rabbit anti-OPN (Novus Biologicals, Littleton, CO, USA); polyclonal rabbit anti-OCN (Novus Biologicals), mitochondrial dynamics antibody sampler kit (Cell Signalling Technology, Danvers, MA, USA); and monoclonal mouse anti-β-actin (Beyotime, Shanghai, China) overnight at 4 ° C. This was followed by incubation with secondary antibodies at 20 °C–25 °C for 1 h. Immunoreactive bands were visualized by chemiluminescence detection reagents (Millipore, Temecula, CA) according to the manufacturer’s instructions. Band intensities were quantified using ImageJ 1.36b (NIH, Bethesda, MD, USA).
4
Western Blot Protein Expression Analysis
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Cells were collected and lysed in RIPA buffer (Beyotime, ShangHai, China) with protease inhibitor on ice for 30 min, followed by centrifugation at 14, 000g for 10 minutes at 4°C. BCA protein assay kit (Pierce, USA) was used to quantify the protein concentration. The protein was diluted in 4× loading buffer (TransGen, BeiJing), and then boiled for 5 min at 100℃. Equal amounts of protein was loaded onto SDS-PAGE (10%) gels for electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked with 5% Albumin Bovine (BSA; Biotechnology, Lot# 0164C419) for 1 hour at room temperature and then incubated with the following primary antibodies at 4℃ overnight: mouse monoclonal anti-β-actin (1:1000; Beyotime, ShangHai), rabbit monoclonal anti-OIP5 (1:200; Sigma, St. Louis, MO USA). After washing 3 times, the membranes were incubated with a secondary antibody (1:5000 dilution; Abcam) for 2 hours. Then, the membranes were visualized after development with an enhanced chemiluminescence (ECL) substrate (Thermo, USA). Chromogenic reaction was performed by Image Lab 5. 2 software (GelBoc, USA). Finally, Adobe Photoshop software(Adobe Systems, USA) was used to analyze the protein bands.
5
Western Blot Analysis of ESR1, DNAJC12, and ERBB4 in MCF-7 Cells
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MCF-7 cells were harvested, and then lysed in RIPA buffer containing PMSF (Solarbio, R0010) and proteinase inhibitors (Sangon Biotech, C600386-0001), and the lysates were clarified by centrifugation at 12,500 rpm for 15 min at 4°C. A BCA Protein Assay Kit (Beyotime Biotechnology, P0012) was used to determine protein concentration. Total cellular protein was separated by SDS-PAGE and electro-transferred onto PVDF membranes (Millipore, ISEQ00010). Then the membranes were blocked with 5% milk powder (Sangon Biotech, A600669-0250) and washed by 0.1% TBS/T for 10 min. Subsequently, the membranes were incubated with primary antibody. The following primary antibodies were used: mouse monoclonal anti-ESR1 (Santa Cruz Biotechnology, sc-8002), rabbit polyclonal anti-DNAJC12 (Proteintech, 12338-1-AP), rabbit polyclonal anti-ERBB4 (Proteintech, 19943-1-AP), or mouse monoclonal anti-β-actin (Beyotime Biotechnology, AF0003) at 4°C overnight. After washing three times in 0.1% TBS/T, membranes were incubated with the secondary HRP-conjugated goat anti-rabbit IgG (H + L) (Beyotime Biotechnology, A0208) or HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime Biotechnology, A0216). Proteins were visualized with an ECL system.
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