Total protein from H9C2 cells was extracted using RIPA buffer (Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors. A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) was used to detect protein concentration. Protein lysates (40 µg per lane) were subjected to 12% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). Membranes were blocked for 1 h at room temperature using 5% skim milk and subsequently incubated with the following primary antibodies at 4˚C overnight: LRH-1 (cat no. 12800; 1:1,000; Cell Signaling Technology, Inc.), cyclin D1 (cat no. 2978; 1:1,000; Cell Signaling Technology, Inc.), β-catenin (cat no. 8480; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat no. 4970; 1:1,000; Cell Signaling Technology, Inc.). The membranes were then incubated with horseradish peroxidase-coupled secondary antibody (anti-rabbit immunoglobulin G, cat no. 7074; 1:1,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Protein bands were visualized with an ECL western blotting kit (EMD Millipore).
Ecl western blotting kit
Manufactured by Merck Group
Sourced in United States, Germany
The ECL Western blotting kit is a laboratory equipment product used for the detection and analysis of proteins in biological samples. It utilizes a chemiluminescent detection method to visualize and quantify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Second antibody, by Abcam (3 mentions)
Pvdf membrane, by Roche (3 mentions)
Chemi image system fusion fx, by Vilber (2 mentions)
Bca assay kit, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
30 protocols using ecl western blotting kit
1
Protein Extraction and Western Blot Analysis of H9C2 Cells
2
Protein Extraction and Western Blot Analysis
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Cellular lysates were obtained by suspending 0.5 × 106 cells in 80 µL of RIPA buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6). The cells were disrupted by vortexing and extracted at 4 °C for 30 min. Protein concentrations were evaluated using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were electrotransferred onto Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). Specific proteins were detected using an ECL Western blotting kit (EMD Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The signal strength was analyzed using a Chemi Image System Fusion FX (Vilber Lourmat, Collégien, France).
3
Western Blot Analysis of THP-1 and FLS Cells
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THP-1 cells (2 × 106 cells/well in 60-mm dishes) and FLS (2 × 106 cells/well in 100-mm dishes) were plated for western blotting analysis. After culturing for 24 h, cells were lysed in RIPA buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6) and supernatant fractions were collected. Protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoresed in SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare Life Science, Pittsburgh, PA, USA). Subsequently, the membranes were incubated with blocking buffer (0.05% Tween 20 with 5% non-fat dry milk in Tris-buffered saline (TBS)) for 1 h and then incubated for 24 h with appropriately diluted primary antibodies against specific target proteins. Next, the membranes were washed with TBS with Tween 20, and then incubated at room temperature for 1 h with the appropriate secondary antibodies. The specific proteins were detected with ECL western blotting kit (EMD Millipore, Darmstadt, Germany) according to the manufacturer’s protocols, and the signal strength was measured using a Chemi Image System Fusion FX (Vilber Lourmat, Collégien, France). Protein bands were quantified using Image-J software. Quantitative analysis of band density using the Image-J is shown as the mean ± standard deviation (SD) of three independent experiments.
4
Western Blot Analysis of Cellular Proteins
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Cellular lysates were extracted using lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethylsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). After being quantified using the BCA Protein Assay Kit (Pierce, Appleton, WI), the proteins samples was separated by SDS-PAGE, electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA, USA), blocked, and incubated with primary antibodies. After being incubated with corresponding secondary antibodies including rabbit IgG-HRP or mouse IgG-HRP(Molecular Probes, Eugene, OR, USA), the blots developed with ECL Western blotting kit (Millipore) according to the manufacturer’s instructions.
5
Profiling the PI3K/Akt/mTOR Pathway in OSCC
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Transfected OSCC cells were lysed using RIPA buffer (Thermo Scientific, Belmont, MA, U.S.A.) at 4°C for 30 min. Proteins in OSCC cells were extracted and separated through 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a PVDF membrane (Roche) via electroblotting. The membranes were blocked in 5% nonfat milk for 1 h at room temperature and then incubated at 4°C overnight with primary antibodies, including anti-PI3K (ab32089), anti-p-PI3K (ab182651), anti-Akt (ab179463), anti-p-Akt (ab38449), anti-mTOR (ab2732), anti-p-mTOR (ab109268), and anti-GAPDH (ab181602) antibodies. Next, the membrane was incubated for 1.5 h at room temperature using second antibody (1:2000, Abcam, China). Target band of protein was visualized using ECL Western blotting kit (Millipore, Boston, MA, U.S.A.).
6
Protein Quantification via Western Blot
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Cells were lysed in cold RIPA buffer, and the protein was separated with 10% SDS‐PAGE, which was then transferred to PVDF membrane (Thermo Fisher, Waltham, MA, USA). After that, the membrane was incubated in PBS with 5% nonfat dried milk (Mengniu, Hohhot, China) for 3 h at 4°C. Then, the membrane was incubated with primary antibodies overnight at 4°C and then with appropriate secondary antibody (Abcam, Cambridge, UK) for 1 h at 37°C. The immune complexes were detected using ECL Western Blotting Kit (Millipore, Boston, MA, USA). The relative protein expression was analyzed using Image‐Pro plus software 6.0 (Media Cybernetics Inc, Rockville, MD), and β‐actin was used as the internal reference.
7
Western Blot Analysis of NR2A, NR2B, and nNOS
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Cultured DRG neurons in 6-well plates were lysed in RIPA buffer (Thermo Scientific, USA) supplemented with protease inhibitors (Thermo Scientific, USA). Debris was sedimented via centrifugation at 14,000 g for 10 min at 4 °C, and the supernatant was collected. The protein concentration was detected using a BCA protein assay kit (Thermo Scientific, USA). Proteins were separated using 8% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, USA). Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [49 (link)] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal). Membranes were rinsed with TBST and incubated with an HRP-conjugated secondary antibody (1:2000; Sigma-Aldrich, USA) for 1 h at room temperature. After washing with TBST, the protein bands were visualized using an ECL Western blotting kit (Millipore, USA). Densitometry was analyzed using AlphaEaseFC software.
8
Protein Expression Analysis in HMECs
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After treatment, HMECs were harvested and homogenized with RIPA lysis buffer (Beyotime Biotechnology) containing protease inhibitor cocktail. Twenty‐five micrograms of isolated protein was subjected to 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Bio‐Rad Laboratories) and then incubated with anti‐STAT3 antibody (1:1,000; CST, Boston, MA), anti‐phospho‐STAT3 antibody (1:1,000; CST), and anti‐phosphatase and tensin homolog (PTEN) antibody (1:1,000; CST) at 4°C overnight. After incubation with the Horseradish peroxidase (HRP)‐conjugated antibody (CST), the membranes were visualized using an ECL Western Blotting Kit (Millipore).
9
Western Blot Protein Quantification
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Cellular lysates were prepared by suspending 1 × 106 cells in 100 μL lysis buffer (137 mM NaCl, 15 mM ethylene glycol tetraacetic acid, 0.1 mM sodium orthovanadate, 15 mM MgCl2, 0.1% Triton X-100, 25 mM 3-(N-morpholino)propanesulfonic acid, 100 μM phenylmethylsulfonyl fluoride, and 20 μM leupeptin, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4 °C for 30 min. The lysates were centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant fractions were collected. Approximately 50 μg of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were electrotransferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (0.05% Tween® 20 with 5% non-fat dry milk in TBS) for 3 h. After washing three times with TBST, the membranes were incubated with primary antibody overnight. Detection of specific proteins was carried out with an ECL Western blotting kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Signal intensity was analyzed using the Chemi Image documentation system (Fusion Rs7, VILBER LOUTMAT, Collégien, France).
10
Protein Extraction and Western Blot Analysis
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The protein of tissues was extracted and separated using 10% sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Roche) by electroblotting. PVDF membrane was blocked by non-fat milk at room temperature for 24 hours or 4 °C for overnight. The membrane was incubated by primary antibodies (1:1000) at 4 °C for overnight, and then incubated with second antibody (1:2000, Abcam, China) for 1.5 hours at room temperature. The target band of protein was analyzed using ECL Western blotting kit (Millipore, Boston, MA).
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