To assess protein overexpression of SEZ6 in H4-SW, protein extracts (18 μg) were separated on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane. Blots were developed using horseradish peroxidase-conjugated secondary antibodies and the ECL chemiluminescence system (MerckMillipore, Burlington, MA, USA). All blots were normalized to α-tubulin and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The following antibodies were used: anti-α-tubulin (1:7500; Abcam, Cambridge, UK) and anti-SEZ6 (1:1000; Aviva Systems Biology, San Diego, CA, USA).
Ecl chemiluminescence system
Manufactured by Merck Group
Sourced in United States, Germany
The ECL chemiluminescence system is a laboratory equipment designed for the detection and quantification of proteins in Western blotting analyses. It utilizes a chemiluminescent reaction to generate light, which is then captured and measured by a detection device. The system provides a sensitive and reliable method for visualizing target proteins in complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protein assay reagent, by Bio-Rad (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Las 3000 mini system, by Fujifilm (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Anti α tubulin, by Abcam (1 mentions)
Automatic microplate reader, by PerkinElmer (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Nitrocellulose membrane, by Beyotime (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ripa cell lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Beyotime (1 mentions)
Ab194697, by Abcam (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions)
Ab194113, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ab32138, by Abcam (1 mentions)
20 protocols using ecl chemiluminescence system
1
Western Blot Analysis of SEZ6 Protein
2
TGF-β1 Protein Expression in Rat Liver
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The liver of SD rats was removed from liquid nitrogen and cut. The mixture was homogenized with 500 µl of RIPA (tissue lysate, Beyotime Biotechnology, Guangzhou, China) and 1% cocktail (protease inhibitor; ProteinTech Group, Inc., Chicago, IL, USA) and centrifuged at 13,000 × g, 4°C for 30 min. The supernatant was measured for its protein concentration using an automatic microplate reader (PerkinElmer, Inc., Waltham, MA, USA). Protein sample (40 µg) was taken and separated with 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The total protein was transferred onto nitrocellulose membrane (NC) and the band at 25 kDa was incubated with TGF-β1 primary antibody (1:1,000, cat. no. E-CL-H0109c; Cell Signaling Technology, Inc., Danvers, MA, USA). The abundance of the target protein was measured under the ECL chemiluminescence system (Merck KGaA, Darmstadt, Germany) using ultra-sensitive chemiluminescent (Merck KGaA) and the grey value was analyzed with ImageJ software (V2.1.4.7; National Institutes of Health, Bethesda, MD, USA).
3
Protein Expression Analysis in A549 Cells
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A549 cells were washed with pre-cold PBS and lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was determined with a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Samples were separated by means of electrophoresis in SDS-PAGE and were then transferred to nitrocellulose membranes and were incubated with the indicated primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. The specific protein bands were detected with the ECL chemiluminescence system (Merck Millipore, Darmstadt, Germany).
4
Western Blot Protein Analysis Protocol
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Cells were harvested and rinsed twice with ice-cold PBS and lysed with RIPA cell lysis buffer (Beyotime, Shanghai, China). The protein concentration was quantified by a BCA protein assay kit (Beyotime, Shanghai, China). Cell lysates containing equal amounts of protein were mixed with 2 × Laemmli buffer (Beyotime, Shanghai, China) and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Merck Millipore, Germany) for immunoblotting. After blocked in 0.5% nonfat dry milk, the nitrocellulose membranes were incubated with the indicated primary antibodies at room temperature for 2 h. After incubation with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Beyotime, Shanghai, China), the Immunoreactive signals were visualized using the ECL chemiluminescence system (Merck Millipore, Germany).
5
Western Blot Analysis of Hepatic Proteins
6
Western Blot Analysis of Cellular Proteins
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Total protein was extracted from cultured cells and tissues. The cells were lysed in RIPA buffer with a protease inhibitor. The 10–15% SDS-PAGE gels were used to separate the lysates, and were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% nonfat milk with Tween for 1 h and incubated with indicated primary antibodies overnight at 4 °C. Then the membranes were incubated with fluorescence-conjugated secondary antibodies (1:5,000, Invitrogen, USA). ECL chemiluminescence system (Merck Millipore, Germany) was used for signal detection. To perform Western blot analysis, the following antibodies were used: rabbit anti-LC3B (ab48394, Abcam, 1:1,000); rabbit anti-PARP (ab32138, Abcam, 1:1,000); rabbit anti-c-Myc (ab32072, Abcam, 1:1,000); rabbit anti-Rab7 (ab137029, Abcam, 1:1,000); rabbit anti-p62 (sc-48402, Santa Cruz, 1:1,000); rabbit anti-HIF-1α (sc-13515, Santa Cruz, 1:1,000); mouse anti-β-actin (sc-58673, Santa Cruz, 1:2,000).
7
Western Blot Analysis of Protein Levels
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Protein levels in H9C2 cell lysates and myocardial tissue homogenates were analyzed by Western blot. BCA protein assay kit was applied to determine protein concentrations. Briefly, 20 µg of protein samples were separated by 10% SDS-PAGE and then electroblotted onto polyvinylidene fluoride membranes. The membrane was blocked in 5% bull serum albumin for 1.5–2 h at room temperature, then washed three times by TBST, and submerged in diluted primary antibodies (1:1,000) overnight at 4°C, followed by the secondary antibodies for 2 h at room temperature. Finally, protein-attached bands were visualized using the ECL chemiluminescence system (Millipore Corp. MA, United States).
8
Caco-2 Cell Stimulation and Protein Analysis
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Caco-2 cells were stimulated with 100 μM ATP for 6 and 18 h. Cells were washed with ice-cold PBS and lysed in Triton buffer (40 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.2 mM sodium orthovanadate, 40 mM glycerophosphate, 0.1 mM phenylmethanesulfonyl fluoride and protease inhibitor mixture from Sigma-Aldrich). Protein concentration was determined using the Bio-Rad Protein Assay reagent. Samples were heated for 5 min at 95°C, subjected to 7% SDS-PAGE and transferred onto polyvinylidene fluoride membranes for protein immunoblotting as previously described [32 (link),33 (link)]. Immunoblotting for MRP2 was performed using a 1/1,000 dilution of rabbit polyclonal anti-MRP2 and the specific protein band was detected using a 1:10,000 dilution of HRP-conjugated donkey anti-rabbit IgG and visualized on autoradiographic film using the Millipore ECL chemiluminescence system. Signal was normalized as described with 1/5,000 dilution of rabbit anti-β-tubulin antibody [32 (link),33 (link)].
9
Protein Expression Analysis in BV2 Cells
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In vitro, BV2 cells were harvested at the indicated days and lysed in ice-old TNE buffer (150 mM NaCl; 10 mM Tris, pH 8.0; 1 mM EDTA; 10% glycerol; 1% NP-40 with phosphatase protease inhibitors). Lysates were centrifuged at 14,000×g at 4 °C for 10 min. Protein lysates were boiled in sample buffer and resolved on 10% SDS-PAGE gels. The following primary antibodies were used in this study: rabbit anti-iNOS (Abcam, ab15323, 1:100), rabbit anti-MCT1 (Millipore, AB3540P, 1:1000), mouse anti-MCT4 (Santa Cruz, sc-376140, 1:200), rabbit anti-PFKFB3 (Proteintech, 13763-1-AP, 1:2000), rabbit anti-STAT1 (Bioworld, BS1333, 1:500), rabbit anti-phospho-STAT1 (CST, 9167, 1:1000), and mouse anti-β-actin (Proteintech, 60008-1-Ig, 1:2000). The appropriate HRP-conjugated second antibodies (1:5000) were from Calbiochem. The blots were reacted with an ECL chemiluminescence system (Millipore) and the gray intensity analysis was performed using the Quantity One software.
10
Western Blot Analysis of Mouse Brain Proteins
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For mouse brain tissue, mice were killed by decapitation, the brain tissues of the prefrontal cortex were obtained from whole brains, and the tissues were homogenated with a sample buffer supplemented with a phosphatase inhibitor and protease inhibitor cocktail on ice and boiled. For cells, cell proteins were extracted in 1x Laemmli lysis buffer and boiled. Protein concentration was determined by using a Bio-Rad protein assay reagent (Hercules, CA, USA). An equal quantity of proteins was separated by 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Burlington, MA, USA). The membranes were incubated with a primary antibody overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. The immunoblots were visualized using an ECL chemiluminescence system (Millipore Corporation, Billerica, MA, USA), and the blots were analyzed using ImageJ software (NIH, Bethesda, MD, USA).
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