Total protein extracts were obtained by grinding 100 mg leaf tissues in protein extraction buffer [20 mM Tris-HCl, pH 7.5, 5 mM ethylenediaminetetraacetic acid, 5 mM ethylene glycol tetraacetic acid, 10 mM dithiothreitol, 0.05% sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride]. The extracts were centrifuged for 10 min at 4°C, and the resulting supernatants were loaded on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels with loading buffer. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane for 2 h. The membrane was then incubated with 1:4000 anti-GFP antibodies (Sigma Sigma-Aldrich, St. Louis, MO, United States) in phosphate-buffered saline (pH 6.9). Horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) was used at 1:5000 dilution and the results were visualized and interpreted using an enhanced chemiluminescence detection system according to the manufacturer’s recommendations (Applygen Technologies Inc., Beijing, China). The intensity of bands on the blots was quantified by densitometry of images using ImageJ software (Media Cybernetics, San Diego, CA, United States).
Enhanced chemiluminescence detection system
Manufactured by Applygen
Sourced in China
The Enhanced Chemiluminescence Detection System is a laboratory instrument used to detect and quantify chemiluminescent signals. It employs a sensitive photon detector to capture light emissions from chemically induced light-producing reactions. The core function of this system is to provide accurate and reliable measurement of these luminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Radioimmunoprecipitation assay lysis buffer, by Beyotime (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Laemmli sample buffer, by Bio-Rad (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Imagej software, by Media Cybernetics (1 mentions)
Anti rabbit immunoglobulin g horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Bca protein assay kit, by Applygen (1 mentions)
Col 1, by Cell Signaling Technology (1 mentions)
Col 3, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
8 protocols using enhanced chemiluminescence detection system
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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Total protein from hHSFs was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) 48 h after transfection. A bicinchoninic acid protein assay was performed to determine the quality of the protein samples. Equal amount of protein (30 µg per lane) were separated by SDS-PAGE (12% gel) and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skimmed milk in Tris buffered saline with 0.1% Tween-20 at room temperature for 1.5 h, followed by incubation with primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) against Smad2 (cat. no. 5339; 1:1,000), cyclin-dependent kinase (CDK)2 (cat. no. 2546; 1:1,000), CDK4 (cat. no. 12790; 1:1,000), apoptosis regulator Bcl-2 (Bcl-2; cat. no. 4223; 1:1,000), apoptosis regulator BAX (Bax; cat. no. 5023; 1:1,000) and β-actin (no. 4970; 1:5,000) at 4°C overnight. Subsequently, the membranes were incubated with anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence detection system (Applygen Technologies, Inc., Beijing, China). ImageJ 1.38X (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometry.
3
Western Blot Analysis of Granulosa Cells
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The collected granulosa cell samples were washed with PBS and lysed in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). The equal amounts of protein (30μg) were electrophoresed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12% acrylamide gel), and proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in tris-buffered saline solution containing 0.1% tween-20 (TBST) at 37 °C for 1h, the membranes were incubated within primary rabbit polyclonal antibodies at 4 °C, overnight. Optimal dilutions for each antibody were determined (β-Actin, 1:2000; LC3, Bim, p-ERK1/2, ERK1/2, 1:1000; Beclin1, 1:500; Bcl-2, 1:1000; Bax, 1:500). Membranes were washed thrice with TBST solution and incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:2000 for 1h. Membranes were washed and protein bands were visualized using an enhanced chemiluminescence detection system (Applygen Technologies Inc., China). Images were taken and protein immunoblots were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Protein Samples
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The cells were lysed in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). An equal amount of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% acrylamide gel), and proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk Tris-buffered saline containing 0.1% Tween-20 (TBST), the membranes were incubated with primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:2000 for 1 h. The protein bands were visualized using an enhanced chemiluminescence detection system (Applygen Technologies Inc., Beijing, China). The western blotting images were processed using Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Protein Detection
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The cells were harvested and lysed with cold cell lysis buffer containing 1% Triton X-100 and 1% protease inhibitor (Beyotime Institute of Biotechnology, USA). Total lysate was separated on a 12% SDS-PAGE gel then transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (10 mM Tris-Cl at pH 7.5 and 150 mM NaCl) containing 0.05% Tween 20 (TBST) at 37 °C for 1 h, and then incubated with primary antibody at room temperature for 4 h. Detection was performed using horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 1 h. Blots were developed using enhanced chemiluminescence detection system (Applygen, China).
6
Western Blot Analysis of MAPK1 and GAPDH
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RIPA lysis buffer (Beyotime Institute of Biotechnology) and protease inhibitor (Beyotime Institute of Biotechnology) were used to extract the proteins from cells. A BCA protein assay was then used to quantify the proteins. Equal amounts of protein (35 µg/lane) were separated via 12% SDS-PAGE and then transferred to PVDF membranes. Subsequently, the membranes were blocked with 5% non-fat milk at room temperature for 90 min and incubated with primary antibodies: MAPK1 (cat. no. ab205819; 1:1,000; Abcam) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. This was followed by incubation with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. Finally, an enhanced chemiluminescence detection system (Applygen Technologies, Inc.) was used to observe the protein bands. For densitometry detection, analysis with ImageJ 1.38X software (National Institutes of Health) was performed.
7
Western Blot Analysis of Signaling Proteins
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Cells were rinsed twice with ice-cold PBS and lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 4 mM EDTA, 0.5% NP-40, 20 mM β-Glycerophosphate, 1 mM NaF with protease inhibitors (Roche, Mannheim, Germany). Protein concentrations were determined using a BCA protein assay kit (Applygen Technologies Inc, Beijing, China). Equal amounts of total protein were loaded and separated by SDS-PAGE gel, and then were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking with 5% BSA at room temperature for 1 h, blots were probed with primary antibodies against P2Y2 (1: 500), β-actin (1: 1000), EGFR (1: 500), ERK1/2 (1: 1000), phospho-EGFR (1: 1000) or phospho-ERK1/2 (1: 1000) at 4 °C overnight. Then the blots were washed with PBS for three times and incubated with secondary antibodies at room temperature for 1 h. The immunoreactive bands were visualized by an enhanced chemiluminescence detection system (Applygen Technologies Inc), and quantified with the software of Quantity One (Bio-Rad).
8
Western Blot Analysis of Smad2, Col I, and Col III
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Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) was used to collect the proteins from cells, and bicinchoninic acid protein assay was then conducted to quantify the protein concentration. Equal amount of protein (40 µg/lane) was separated by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Next, the membranes were blocked with 5% non-fat milk at room temperature for 1.5 h, incubated with primary antibodies: Smad2 (1:1,000; cat. no. 5339; Cell Signaling Technology, Inc.), Col I (1:1,000; cat. no. ab34710; Abcam), Col III (1:1,000; cat. no. Ab7778; Abcam) and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) at 4˚C overnight. Subsequently, membranes were incubated with anti-rabbit horseradish peroxidase-conjugated immunoglobulin G secondary antibody (cat. no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. At the end of the experiment, an enhanced chemiluminescence detection system (Applygen Technologies, Inc., Beijing, China) was used to observe the protein bands. For densitometry detection, analysis with ImageJ 1.38X software (National Institutes of Health, Bethesda, MD, USA) was performed.
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