Western blotting was performed on cancer and healthy samples. The tissues were stored at −80°C. Total protein was extracted by ristocetin-induced platelet aggregation (Beyotime Institute of Biotechnology, Jiangsu, China) in the presence of phosphatase and protease inhibitors according to the manufacturer's instructions (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instructions (Boster Biological Technology Co., Ltd., Wuhan, China). Thirty micrograms of protein per lane were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to 0.45 µm nitrocellulose filter membrane (EMD Millipore, Billerica, MA, USA), followed by blocking with skim milk at room temperature for 2 h. Subsequently, the membranes were incubated with mouse anti-human CLIC1 antibody (ab77214; 1:750 dilution; Abcam, Cambridge, UK) or mouse anti-human β-actin (1:2,000 dilution; Boster Biological Technology, Pleasanton, CA, USA) overnight at 4°C. The membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:2,500 dilution; Boster Biological Technology) for 1 h and then subjected to semi-quantification by electrochemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Mouse anti human β actin
Manufactured by Boster Bio
Sourced in China, United States
Mouse anti-human β-actin is a primary antibody that specifically binds to the β-actin protein, which is a ubiquitous and highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody can be used for the detection and quantification of β-actin in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Boster Bio (1 mentions)
Phosphatase and protease inhibitors, by Solarbio (1 mentions)
Rabbit anti human traf4 antibody, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Boster Bio (1 mentions)
Goat anti mouse igg hrp, by Boster Bio (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Bca assay, by Beyotime (1 mentions)
Protease inhibitor mix, by Beyotime (1 mentions)
Ecl western blot kit, by CWBIO (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Rabbit anti human cyclin e, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Western blotting luminol reagent, by Tiangen Biotech (1 mentions)
Donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
6 protocols using mouse anti human β actin
1
Quantifying CLIC1 Protein in Cancer Samples
2
Western Blotting of Apoptosis Markers
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Western blotting was performed as described previously6 (link). Rabbit anti-human TRAF4 antibody and mouse anti-human MDM2 and p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human p-AKT (phospho S473), AKT, p-MDM2 (Phospho Ser166), Bax, p-GSK-3β (phospho S9), GSK-3β, cyclin D1, and c-Myc antibodies were purchased from CST (Boston, MA, USA). Mouse anti-human β-actin and GAPDH antibodies were purchased from Boster (Wuhan, China).
3
Western Blot Analysis of TNFR1 and TNFR2
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Briefly, total proteins were extracted from the cells using RIPA lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.4) and protease inhibitor mix, (all from Beyotime Institute of Biotechnology, Jiangsu, China) and an ultrasonic cell disruptor (Branson Instrument Co., Danbury, CT, USA). The protein concentrations were determined by BCA assay (Beyotime Institute of Biotechnology, Jiangsu, China). Equal protein samples (30-40 μg) were separated on 10% SDS-PAGE gels (Beyotime Institute of Biotechnology, Jiangsu, China) and transferred onto the polyvinylidene difluoride membranes (Millipore, USA). Membranes were incubated with primary antibodies: rabbit anti-human TNFR1 (1 : 1000), rabbit anti-human TNFR2 (1 : 10 000; both from Epitomics, Abcam, Burlingame, CA, USA), and mouse anti-human β-actin (1 : 1000, Boster, Wuhan, China). Secondary antibodies were goat anti-mouse IgG-HRP (1 : 1000) and goat anti-rabbit IgG-HRP (1 : 1000; both from Boster, Wuhan, China). Images were obtained by chemiluminescence using an ECL-Western blot kit (CW Biotech, Beijing, China) and analyzed using ImageJ software (1.44p, USA).
4
Western Blot Analysis of EMF-Induced Protein Expression
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Western blot analysis was performed as described by a previous study[16 (link)]. After 50 Hz-EMF exposure (1 mT, 12 h), cells were harvested and lysed in RIPA buffer (Beyotime, China) to obtain protein samples. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). Then, the proteins (30 μg/well) were subjected to 10% SDS-PAGE. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad, USA), blocked and incubated with various primary antibodies at 4°C overnight. Mouse anti-human β-actin (1:1000, Boster, China), rabbit anti-human Cyclin E (1:1000, Santa Cruz, USA), and rabbit anti-human Cyclin D1 were used as primary antibodies. The membranes were washed and incubated with secondary antibodies and then detected by an ECL kit (Millipore, USA).
5
Analyzing TCR Subunits in HEK293 Cells
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HEK 293 cells were harvested 24 hours after transfection, mixed with RIPA lysis buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 10 mmol/L phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, and 100 mmol/L sodium orthovanadate), and incubated on ice for 30 min to isolate total proteins. Proteins (100 μg) were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes (Invitrogen, USA) using a damp-dry transfer device (Bio-rad, USA). After blocking for 1 hours in 5% defatted milk powder in PBS, the membranes were washed and probed with a mouse anti-human TCR β monoclonal antibody and a rabbit anti-human TCRα polyclonal antibody (Santa Cruz, USA). Similar studies were performed with 1:500 mouse-anti-human β-actin (BOSTER, Wuhan, China). The antibodies were detected using 1:10,000 horseradish peroxidase-conjugated, goat-anti-rabbit IgG and donkey-anti-mouse IgG (Jackson ImmunoResearch, USA). A Western blotting luminol reagent (Tiangen, Beijing, China) was used to visualize bands corresponding to each antibody.
6
Geranylgeranylacetone Modulates Apoptosis
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Geranylgeranylacetone (GGA) was obtained from Eisai China (Shanghai, China). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kits (fluorescent), annexin V fluorescein isothiocyanate apoptosis detection kits and protease inhibitors were obtained from Calbiochem (San Diego, CA, USA). In addition, the following antibodies were used: mouse anti-human HSP72 (1:1,000; Stressgen Biotechnologies, Victoria, BC, Canada), rabbit anti-human XIAP (1:1,000; BD Biosciences, San Jose, CA, USA), mouse anti-human Smac/DIABLO (1:1,000; BD Biosciences), rabbit anti human pro caspase 3 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and mouse anti-human β-actin (1:2,000; Boster, Wuhan, China). Horseradish peroxidase-conjugated anti-mouse IgG and horseradish peroxidase conjugated anti-rabbit IgG were obtained from Jackson ImmunoResearch (West Grove, PA, USA). All remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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