The frozen tissue samples were sectioned to 7 μm slices for protein preparation by the method described elsewhere.22 The sample proteins (50 µg/well) were separated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.5% Tween 20) at 4°C, rinsed 10 mins for three times with TBS-T, followed by 3 h incubation at room temperature with the first antibody and then 1 h incubation with HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed Lab, Inc, USA). The bound antibody was detected using the enhanced chemiluminescence system (Roche GmbH, Mannheim, Germany). After removing the labeling signal by incubation with stripping buffer, the membrane was re-incubated with other antibodies one by one until all of the parameters (ARHI, PIAS3 and p-STAT3) were examined with the same antibodies used in immunohistochemical staining. β-actin was cited as the quantitative control.
Enhanced chemiluminescence system
Manufactured by Roche
Sourced in Germany
The Enhanced Chemiluminescence System is a laboratory equipment used to detect and quantify proteins in Western blotting experiments. It utilizes a chemiluminescent substrate to generate a light signal proportional to the amount of target protein present in the sample. This system enables sensitive and reliable protein detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Cytiva (10 mentions)
Horseradish peroxidase conjugated anti rat igg, by Zymo Research (3 mentions)
Tween 20, by Merck Group (2 mentions)
Uvp biospectrum imaging system, by Analytik Jena (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Hrp conjugated anti rabbit or anti mouse igg, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti clusterin, by Santa Cruz Biotechnology (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
Horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
X ray film, by Kodak (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Fabp5, by Proteintech (1 mentions)
Rar β, by Bioss Antibodies (1 mentions)
Ppar β δ, by Bioss Antibodies (1 mentions)
Crabp2, by Proteintech (1 mentions)
Hrp labeled secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
20 protocols using enhanced chemiluminescence system
1
Protein Analysis of Frozen Tissue Samples
2
Western blot analysis of protein expression
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Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China). The sample proteins (50 μg/well) were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris–HCl, pH 8.0, 150 mM NaCl and 0.5% Tween 20) at 4°C, and then rinsed 10 min for three times with TBS-T, followed by incubation for 3 h at room temperature with the first antibody in appropriate concentrations (MGMT: 1:1000; STAT3: 1:800; p-STAT3: 1:800; Bcl-2: 1:800; survivin: 1:800; all from Santa Cruz Biotech, Inc., Santa Cruz, CA, USA), and then incubation with HRP conjugated anti-mouse or anti-rabbit IgG (Proteintech, Chicago, IL, USA) 1 h. The bound antibody was detected using the enhanced chemiluminescence system (Roche GmbH, Mannheim, Germany). After removing the labeling signal by incubation with stripping buffer,16 (link),17 (link) the membrane was reprobed with other antibodies one by one until all of the parameters were examined. Image J was used to measure the density of bands (National Institutes of Health, Bethesda, MD).
3
Western Blot Analysis of Cellular Proteins
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Total cellular proteins were prepared from the cells under different culture conditions [41 (link)]. The sample proteins (15μg/well) were separated by electrophoresis in 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris–Cl, pH 8.0, 150 mM NaCl and 0.5% Tween 20) at 4°C overnight, rinsed three times with TBS-T and followed by 3h incubation at room temperature with the first antibody, and 1h incubation with HRP-conjugated anti-rabbit IgG (Zymed Lab Inc., San Francisco, CA, USA). The bound antibody was detected using the enhanced chemiluminescence system (Roche, Penzberg, Germany). After removing the labeling signal by incubation with stripping buffer (62.5 mM Tris–HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS) at 55°C for 30 min, the membrane was reprobed with other antibodies one-by-one until all of the parameters were examined.
4
Western Blotting of Tumor Cell Lysates
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Samples containing equal amounts of protein (depending on the antibody, 5-50μg) from lysates of cultured tumor OS cell lines underwent electrophoresis on SDS-polyacrylamide gel and were transferred to nitrocellulose filters. The filters were blocked in PBS containing 3% BSA and 0.1% Tween at room temperature for 1h and blots were probed overnight at 4°C with primary antibodies (anti-clusterin, Santa Cruz Biotechnology, Dallas, TX, USA; anti-vinculin, Sigma-Aldrich Corp, St. Louis, MO, USA) to detect proteins of interest. After incubation, the filters were washed 3 times with washing buffer (PBS containing 0.1% Tween) for 5min. Filters were then probed with the secondary antibody coupled to horseradish peroxidase. Antibody binding was visualized with the enhanced chemiluminescence system (Roche Molecular Biomedicals).
5
Protein Extraction and Western Blotting from Liver Tissue
6
Western Blot Protocol for Protein Analysis
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The DNA-transfected cells or virus-infected cells were harvested with phosphate-buffered saline (PBS) and total cell extracts were prepared by boiling the cell pellets in sodium dodecyl sulfate (SDS) loading buffer. Equal amounts of the clarified cell extracts were separated by SDS-PAGE, and then transferred onto a nitrocellulose membrane (GE Healthcare Life Sciences) or PVDF membrane (Millipore). The membrane was blocked for 1 h in PBST (PBS plus 0.1% Tween 20 [Sigma]) containing 5% skim milk and then washed with PBST. After incubation with the appropriate antibodies, the proteins were visualized by standard procedures using an enhanced chemiluminescence system (Roche) and x-ray film (Kodak).
7
Western Blotting of Thyroid Proteins
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For Western blotting, total cellular proteins were prepared from the thyroid tissues by the method described previously [27 (link)]. Fifty micrograms of sample protein was separated with 12% SDS/PAGE, and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in NaCl/Tris-T (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) at 4 °C overnight. It was rinsed three times (10 min each) with NaCl/Tris-T, and this was followed by 3 h of incubation with the same first antibodies that were used in immunohistochemical staining in the appropriate concentrations (IL-6, 1:500; COX-2, 1:500; NF-κB/p65, 1:500; β-actin, 1:3000, and IkBα, 1:1000) and 1 h of incubation with horseradish peroxidase-conjugated anti-rat IgG (Zymed Laboratories, San Francisco, CA, USA). Immunolabeling was detected with an enhanced chemiluminescence system (Roche, Mannheim, Germany), and visualized with the UVP Bio-spectrum Imaging System (UVP, Upland, CA, USA). β-actin was used as the internal quantitative control in densitometry analyses.
8
Western Blot Analysis of Proteins
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Total cellular proteins were prepared from the cells under different culture conditions. The sample proteins (50 μg/well) were separated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris-HCl, pH8.0, 150 mM NaCl and 0.5% Tween 20) at 4 °C, rinsed 10 min for three times with TBS-T, followed by 3 h incubation at room temperature with the first antibody and then 1 h incubation with HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed Lab, Inc). The bound antibody was detected using the enhanced chemiluminescence system (Roche GmbH, Mannheim, Germany). After removing the labeling signal by incubation with stripping buffer,8 (link) the membrane was reprobed with other antibodies one by one until all of the parameters were examined.
9
Western Blot Analysis of Cellular Proteins
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Total cellular proteins were prepared from the cells under different culture conditions. The sample proteins (50 μg/well) were separated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM Tris–HCl, pH8.0, 150 mM NaCl and 0.5% Tween 20) at 4°C, rinsed 10 minutes for three times with TBS-T, followed by 3 h incubation at room temperature with the first antibody in appropriate concentrations (Notch1: 1:800; Notch2: 1:800; Hes1: 1:2500; Wnt2: 1:800; β-catenin: 1:800; E-cadherin: 1:600; STAT3: 1:800; Bcl-2: 1:800; c-Myc: 1:600; survivin: 1:800), and then 1 h incubation with HRP-conjugated anti-mouse or anti-rabbit IgG (Zymed Lab, Inc). The bound antibody was detected using the enhanced chemiluminescence system (Roche GmbH, Mannheim, Germany). After removing the labeling signal by incubation with stripping buffer [8 (link)], the membrane was reprobed with other antibodies one by one until all of the parameters were examined.
10
Western Blot Protein Detection
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Cells were washed with cold PBS and then placed in 1X sample buffer. Cell lysates were prepared by boiling, separated by SDS-PAGE, and then transferred onto nitrocellulose membranes (GE Healthcare). Membranes were blocked for at least 1 h in PBST (PBS plus 0.1% Tween 20 [Sigma]) containing 5% skim milk and then washed with PBST. After incubation with appropriate antibodies, proteins were visualized using an enhanced chemiluminescence system (Roche).
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