Cells were washed and lysed in ice-cold RIPA buffer (50 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM of ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), and 1 mM of EDTA) with a protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN, USA). Then, 50 μg of the total proteins was separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). Afterward, the membranes were blocked with 5% nonfat milk for 1 hour at room temperature, incubated with primary antibodies overnight at 4°C, and incubated with the appropriate horseradish peroxidase–conjugated secondary antibody (Promega Corporation, Madison, WI, USA) for 1 hour. Membrane blot signals were detected using the enhanced chemiluminiscence detection kit (Amersham Biosciences).
Horseradish peroxidase conjugated secondary antibody
Manufactured by Promega
Sourced in United States, France
Horseradish peroxidase-conjugated secondary antibodies are laboratory reagents used to detect and quantify specific target proteins in various analytical techniques, such as Western blotting and ELISA. They consist of secondary antibodies that are chemically conjugated to the enzyme horseradish peroxidase, which catalyzes a colorimetric or chemiluminescent reaction to signal the presence of the target.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (4 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Bradford assay, by Bio-Rad (4 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Extrapage one precast gels, by Nacalai Tesque (3 mentions)
Ecl kit, by Bio-Rad (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
P stat5 y694, by Cell Signaling Technology (3 mentions)
Ps6 s235 236, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Pakt s473, by Cell Signaling Technology (3 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ecl western blotting system, by Promega (2 mentions)
79 protocols using horseradish peroxidase conjugated secondary antibody
1
Western Blot Protocol for Protein Analysis
2
Western Blot Analysis of AKT, FOXO1, and Apoptosis Proteins
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Western blot analysis was conducted as previously described.25 (link) Briefly, protein extracts from harvested experimental cells were prepared using a lysis buffer from Beyotime Biotechnology (Shanghai, China). Thirty micrograms of protein extracts were loaded to a 12% SDS-PAGE, electrophoretically separated and transferred to polyvinylidene membranes (Beyotime Biotechnology, Shanghai, China). After incubating the membrane with 5% non-fat milk blocking buffer at room temperature for 1 hr, the membrane was incubated with a primary antibody overnight at 4 °C. After the incubation with a horseradish peroxidase-conjugated secondary antibody (1:2500, Promega, Madison, WI, USA) at room temperature for 1 hr, the signal was obtained by using an ECL Western blotting system (Promega, Madison, WI, USA), visualized and quantified using the Bio-Rad ChemiDoc MP system. The primary antibodies against AKT (catalog # 4691p, 1:1000), phospho-AKT(Ser457) (catalog # 4060p, 1:2000), FOXO1 (catalog # 2880p, 1:1000) and phospho-FOXO1 (Ser256) (catalog # 9461p, 1:1000) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies specifically against BAX, BCL-2, caspase-3, caspase-8, poly (ADP-ribose) polymerases (PARP) were obtained from Abcam (Abcam, London, UK). β-actin (Sigma Chemical Co., St. Louis, MO, USA) was used as the loading control.
3
Western Blot Analysis of Protein Biomarkers
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For Western blot analysis, total protein extract was isolated from tissue samples by RNA/DNA/Protein Purification Kit (Norgen, CA) as described by manufacturer's instructions. Equivalent amounts of protein extract were separated by electrophoresis on 12% SDS-PAGE gels and blotted onto nitrocellulose. The membranes were blocked with 5% nonfat dry milk and 0.1% Tween-20 in Phosphate-buffered saline and then incubated with antibodies followed by appropriate horseradish peroxidase-conjugated secondary antibody (Promega, USA). Rabbit polyclonal anti-LMTK3 (Abcam, UK) was used diluted 1:500, rabbit polyclonal anti-TGFBI (Thermo Scientific, USA) was diluted 1:1000, rabbit polyclonal anti-SOCS2 (Thermo Scientific, USA) was diluted 1:200. Rabbit polyclonal anti-β-actin (Sigma, USA) diluted 1:400 was used to reveal β-actin as a loading control.
4
Western Blot Protein Detection Protocol
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Harvested cells were resuspended in the lysis buffer (0.5% Nonide P-40, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl) on ice. One-quarter volume of 5×SDS (Sodium Dodecyl Sulfate) sample loading buffer (the buffer contained 100 mM Tris-HCl, 2% beta-mercaptoethanol, 4% SDS, 20% glycerol and 0.02% bromphenol blue, and its pH value was 6.8) was added, followed by boiling for 10–15 minutes. Proteins separated by SDS/PAGE were transferred to a nitrocellulose transfer membrane (GE Healthcare, USA). The membrane was blocked with 5% nonfat dry milk in TBST (the Tris-buffered saline contained 0.1% Tween-20), incubated overnight at 4°C with a primary antibody at 1:1000 dilution, washed 4 times (8 minutes per wash) with TBST, then incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Promega, Madison, WI, USA) for one hour at room temperature, washed extensively with PBS, and finally visualized by enhanced chemiluminescence (ECL; Thermo Fisher Scientific Inc., Waltham, MA, USA).
5
Western Blot Analysis of CORO2A Protein
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Western blot analysis was conducted as previously described (14 (link)). Briefly, protein extracts from harvested experimental cells were prepared using lysis buffer from Beyotime Biotechnology (Shanghai, China). Thirty micrograms of protein extracts were loaded onto a 12% SDS-PAGE gel, electrophoretically separated, and transferred to polyvinylidene membranes (Beyotime Biotechnology, Shanghai, China). After incubating the membrane with 5% non-fat milk blocking buffer at room temperature for 1 h, the membranes were incubated with primary antibody overnight at 4°C. After incubation with a horseradish peroxidase-conjugated secondary antibody (1:2,500, Promega, Madison, WI, USA) at room temperature for 1 h, the signal was obtained by using an ECL Western blotting system (Promega, Madison, WI, USA) and visualized and quantified using the Bio-Rad ChemiDoc MP system. The primary antibodies against CORO2A (Sigma, catalog # HPA041302, 1:1,000) were obtained from Sigma (Sigma Chemical Co., St. Louis, MO, USA). β-actin, also obtained from Sigma, was used as the loading control.
6
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Cells were lysed in cold RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40) containing protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) on ice for 30 min. Lysates were centrifuged at 14,000 rpm at 4°C for 30 min. Protein concentration of the harvested supernatants was quantified with a BCA kit (Beyotime). The proteins were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Burlington, MA, USA). After being blocked with 5% skim milk powder, the membranes were probed with specific primary antibodies at 4°C overnight. Following three washes in TBS solution (Solarbio, China), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:10000, Promega, USA) for 1 h at ambient temperature. Immunocomplexes were visualized using an ECL kit (Tiangen, China) and quantified using the ImageJ software. The antibodies included anti-GAPDH (Proteintech, China; internal control), anti-vimentin (Abcam, Cambrige, UK), anti-BRD4 (Cell Signaling Technology, USA), anti-N-cadherin (Abcam), and anti-E-cadherin (Abcam).
7
Western Blot Analysis of Fks1 Levels
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To examine Fks1 levels, whole-cell protein extracts were prepared as described previously [43 (link)]. Briefly, cells were pelleted, treated with a 1 mL N/β solution (0.25 N NaOH, 1% β-mercaptoethanol), and incubated on ice for 10 min. Then, 100 μL of 100% trichloroacetic acid was added, and incubated on ice for 10 min. The supernatant was discarded after centrifugation at 15,000× g for 2 min at 4 °C. After the pellet was washed with 500 μL of 1 M Tris-HCl (pH 8.0), the cells were resuspended in sodium dodecyl sulfate (SDS) sample buffer including 62.5 mM Tris-HCl (pH 6.8), glycerol (10%), SDS (2%), β-mercaptoethanol (2%), and bromophenol blue (0.005%), incubated for 10 min at 37 °C, and pelleted. Next, the supernatants were loaded in a mini-gel (8%; Bio-Rad, Bio-Rad Laboratories, Inc., Hercules, CA, USA), and Western blotting was performed with mouse monoclonal antibody to Fksl (T2B8) [44 (link)]. Horseradish peroxidase-conjugated secondary antibody was obtained from Promega (Cat. # W4021) and proteins were detected with an enhanced chemiluminescence system (Amersham, ECL Plus). Grayscale analysis was performed using ImageJ.
8
Western Blot Analysis of Kidney Injury Markers
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Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer, and 40 μg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, UK). The membranes were incubated with antibodies against NGAL (Abcam, USA), KIM-1, cleaved CASPASE-3, BAX, BCL-2, cleaved PARP-1, p-NF-κB and NF-κB (all from Cell Signaling Technology, USA) as well as GAPDH (Ambion, USA) and then with a horseradish peroxidase-conjugated secondary antibody (Promega, USA). Visualization of the bands was achieved using the enhanced chemiluminescence western blot analysis system (Pierce, USA). The relative densities of the detected proteins were normalized to that of NF-κB or GAPDH in the same blot.
9
Protein Extraction and Western Blot Analysis
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The total protein of astrocytes was extracted with RIPA buffer and protease inhibitors cocktail from Fisher Scientific, Pittsburgh, PA. After 4–15% gradient SDS-PAGE, the proteins were electro-transferred to Immobilon-P, PVDF membrane (Millipore Corp., Bedford, MA). Nonspecific protein binding sites on the membrane were blocked with nonfat dry milk. Primary monoclonal antibody of Cas9 from Fisher scientific and primary antibody of GAPDH from Sigma (St. Louis, MO) were used. After washing, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Promega, Madison, WI). Blots were washed again and Supersignal West Pico chemiluminescence kit (Thermo Scientific, Waltham, MA) was used to detect target proteins. A BioRad ChemiDoc MP imaging system with Image Lab software was used to catch the images.
10
Immunoprecipitation and Western Blotting
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Yeast cells from the yeast two-hybrid assay were lysed with 1% Triton X-100 containing buffer and glass beads. The lysed cells were then incubated with protein-Sepharose beads coupled to a myc-antibody. Lysates (20 μg) and the immunoprecipitated beads were electrophorized on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane by electroblotting and probed with an HA antibody. Blots were exposed to the corresponding horseradish peroxidase-conjugated secondary antibody (Promega) and developed by enhanced chemiluminiscent staining using ECL western blotting system (Amersham Biosciences). Low-range molecular weight standards were used as reference proteins (Bio-Rad Laboratories).
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