Western blotting was performed according to the published method (9 (link), 12 (link)). Samples (50 µg total protein/lane) were loaded onto SDS-PAGE gels in this experiment. The primary (rabbit anti-human LC3, rabbit anti-human Beclin-1 and rabbit anti-human β-actin antibody) and secondary (anti-rabbit horseradish peroxidase-labeled antibody) antibodies were purchased from Cell Signaling Technology and used at a dilution of 1:1,000 and 1:2,000, respectively. The images were obtained using a CanoScan LiDE 100 scanner (Canon). The results were quantified using Image-J software.
Rabbit anti human β actin antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit anti-human β-actin antibody is a primary antibody that specifically binds to the β-actin protein. β-actin is a highly conserved cytoskeletal protein that plays a crucial role in various cellular processes. This antibody can be used to detect and quantify β-actin expression in human samples, enabling researchers to study its cellular functions and dynamics.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated hrp anti rabbit antibodies, by Santa Cruz Biotechnology (8 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (5 mentions)
Gel pro analyzer, by Media Cybernetics (4 mentions)
Non fat dry milk, by Bio-Rad (4 mentions)
Pvdf membrane, by GE Healthcare (4 mentions)
Bca protein assay kit, by Promega (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti human beclin 1, by Cell Signaling Technology (1 mentions)
Rabbit anti human lc3, by Cell Signaling Technology (1 mentions)
Anti rabbit horseradish peroxidase labeled antibody, by Cell Signaling Technology (1 mentions)
Canoscan lide 100 scanner, by Canon (1 mentions)
Rabbit anti human phospho erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti human p53 antibody, by Cell Signaling Technology (1 mentions)
P38 mapk antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti human e cadherin antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride pvdf membrane, by GE Healthcare (1 mentions)
Mini protean tgx 10 precast gel, by Bio-Rad (1 mentions)
Mouse antihuman tubb1 antibody, by OriGene (1 mentions)
15 protocols using rabbit anti human β actin antibody
1
Western Blotting for LC3, Beclin-1, and β-actin
2
Western Blot Analysis of Protein Expression
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The primary antibodies were rabbit anti-human p53 antibody (#9282T, 1:1000; Cell Signaling Technology, Beverly, MA, USA), p38-MAPK antibody (#8690T, 1:1000; Cell Signaling Technology), rabbit anti-human phospho-ERK1/2 antibody (#4695T, 1:1000; Cell Signaling Technology), and rabbit anti-human β-actin antibody (#4970T, 1:1000; Cell Signaling Technology). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the secondary antibodies. Cell lysates in 1× SDS loading buffer (60 mM Tris–HCl, pH 6.8; 2% SDS; 20 % glycerol; 0.25 % bromophenol blue; and 1.25 % 2-mercaptoethanol) were incubated at 100°C for 10 min to facilitate sample loading for conventional western blotting analysis. The relative protein levels were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).
3
Western Blot Analysis of E-cadherin and β-actin
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The primary antibodies were rabbit anti-human E-cadherin antibody (#3195; 1:1,000) and rabbit anti-human β-actin antibody (#4967; 1:1,000) (both from Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the secondary antibodies. Cell lysates in 1X SDS loading buffer (60 mM Tris-HCl, pH 6.8; 2% SDS; 20% glycerol; 0.25% bromophenol blue; and 1.25% 2-mercaptoethanol) were incubated at 100°C for 10 min to facilitate sample loading for conventional western blot analysis. The relative protein levels were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).
4
Western Blot Analysis of SMAD4 Protein
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The primary antibodies used for western blotting were rabbit anti-human SMAD4 antibody (1:1000; Cell Signaling Technology) and rabbit anti-human β-actin antibody (1:1000; Cell Signaling Technology). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Then, the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk (Bio-Rad, CA, USA) in tri-buffered saline plus Tween (TBS-T) buffer for 2 h. Blots were immunostained with primary antibody at 4°C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualised by using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Bedford, MA, USA). Protein levels were normalized to β-actin.
5
Western Blot Analysis of TS Protein
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The primary antibodies used for western blotting were rabbit anti-human TS antibody (1:1000; Novas) and rabbit anti-human β-actin antibody (1:1000; Cell Signaling Technology). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Blots were immunostained with primary antibody at 4°C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualised by using ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore). Protein levels were normalized to β-actin.
6
Western Blot Analysis of Protein Lysates
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Whole protein lysate was isolated using RIPA buffer [150 mMNaCl, 50 mMTris-HCl (pH = 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] completed with 100X protease/phosphatase inhibitor cocktail (Thermo Fisher, Massachusetts, USA), and protein concentration was measured by BCA protein assay kit (Thermo Fisher). Each lysate from different samples was separated by using Mini-PROTEAN TGX 10% precast gel (Bio-Rad, California, USA) and transferred to nitrocellulose membrane using Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% BSA (Sigma–Aldrich) in TBS-T and then incubated with a mouse antihuman TUBB1 antibody (OriGene, TA506654; 1:3000 dilution) at 4 °C overnight, an anti-Flag HRP antibody (Abcam, ab49763; 1:1000 dilution, Cambridge, UK) at room temperature for 1 h, a rabbit antihuman GAPDH antibody (Cell Signaling, 5174; 3:10000 dilution) at room temperature for 1 h, or a rabbit antihuman β-actin antibody (Cell Signaling, 8457; 1:1000 dilution) at room temperature for 1 h. Upon washing with TBS-T, membranes were incubated with compatible HRP-conjugated antimouse IgG (Cell Signaling, 7076; 1:5000 dilution) or antirabbit IgG (Cell Signaling, 7074; 1:5000 dilution) secondary antibodies at room temperature for 1 h. Protein bands were detected using ECL Plus Western blotting substrate (Thermo Fisher) and ChemiDoc XRS+ imaging system (Bio-Rad).
7
Notch1, NF-κB Pathway Activation in Breast Cancer
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The breast cancer cells were lysed with RIPA buffer with protease inhibitors (Sigma-Aldrich). Protein quantification was carried out using a BCA protein assay kit (Promega). The primary antibodies used for western blot analysis were as follows: Rabbit anti-human Notch1 antibody (#sc6014; 1:500; Santa Cruz Biotechnology), anti-p65-nuclear factor-κB (NF-κB) antibody (#8242; 1:1,000), anti-p50-NF-κB antibody (#3035; 1:1,000) and rabbit anti-human β-actin antibody (#4967, 1:1,000) (all from Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (#e62238, 1:5,000; Santa Cruz Biotechnology) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were then blocked with 5% (5 g/100 ml) non-fat dry milk (Bio-Rad, Hercules, CA, USA) in Tris-buffered saline plus Tween (TBS-T) buffer for 2 h. The blots were immunostained with primary antibody at 4°C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). Protein levels were normalized to β-actin.
8
Western Blot Analysis of TS and β-Actin
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The primary antibodies used for western blot were rabbit anti-human TS antibody (1:1000; Cell Signaling Technology) and rabbit anti-human β-actin antibody (1:1000; Cell Signaling Technology). Horseradish peroxidase(HRP)-conjugated anti-rabbit antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the secondary antibodies. Cell lysates in 1× SDS loading buffer (60 mM Tris–HCl, pH 6.8; 2% SDS; 20% glycerol; 0.25% bromophenol blue; and 1.25% 2-mercaptoethanol) were incubated at 100°C for 10 min to facilitate sample loading for conventional western blotting analysis. The relative protein levels were quantified using densitometry with a Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).
9
Quantitative Analysis of EZH2 and E-cadherin in Osteosarcoma Cells
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Osteosarcoma cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease inhibitors (Sigma-Aldrich). Protein quantification was done using a BCA protein assay kit (Promega). The primary antibodies used for western blotting were rabbit anti-human EZH2 antibody (#4905S, 1:1000; Cell Signaling Technology), rabbit anti-human β-actin antibody (#4967S, 1:1000, Cell Signaling Technology), and rabbit anti-human E-cadherin antibody (#sc-21791, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Horseradish peroxidase-conjugated (HRP) anti-rabbit antibodies (#sc-2004, 1:5000; Santa Cruz Biotechnology) were used as the secondary antibodies. A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). Then, the membrane was blocked with 5% (5 g/100 mL) nonfat dry milk (Bio-Rad, CA, USA) in tri-buffered saline plus Tween (TBS-T) buffer for 2 h. Blots were immunostained with primary antibody at 4 °C overnight and with secondary antibody at room temperature for 1 h. Immunoblots were visualized using Immobilon™ Western Chemiluminescent HRP Substrate (Millipore) and calculated with Image J 1.47 software. Protein levels were normalized to β-actin.
10
Antibody Reagents for NF-κB Pathway Analysis
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Rabbit anti-human phospho-IκBα antibody, rabbit anti-human β-actin antibody, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Cell signaling Technology (Beverly, MA). Rabbit anti-human NF-κB p65 antibody, rabbit anti-human p27Kip1 antibody, rabbit anti-human cyclin D1, rabbit anti-human cyclin D2, rabbit anti-human cyclin D3 antibody, rabbit anti-human cyclin E antibody, and rabbit anti-human ERα antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The synthetic IKKβ inhibitor IMD-0354 (molecular weight, 384.1) was synthesized and kindly provided by the Institute of Medical Molecular Design Inc. (Tokyo, Japan)31 (link). Unless otherwise indicated, all chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO).
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