Western blotting was performed as previously described (25 (link)). Cell lysis was performed in a buffer containing 0.25 M Tris-HCl, 20% glycerol, 4% sodium dodecyl sulfate (SDS) and 10% mercaptoethanol (pH 6.8) supplemented with protease and phosphatase inhibitors. The protein contents were measured using the Micro BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (10 µg) were separated on 10-12% SDS-polyacrylamide gels and electro-transferred onto polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature for 1 h, the membranes were incubated with primary antibodies (1:3,000; collagen-II, cat. no. ab34712, Abcam; aggrecan, cat. no. ab194594, Abcam; MMP-2, cat. no. ab97779, Abcam; GAPDH, cat. no. ab9485, Abcam) in TBST containing 5% non-fat milk overnight at 4°C. Secondary horseradish peroxidase‑conjugated antibodies (1:6,000; Goat Anti-Rabbit IgG H&L, cat. no. ab6721; Goat Anti-Mouse IgG H&L, cat. no. ab205719, Abcam) were added at room temperature for 1 h, and immunoblots were developed using an enhanced chemiluminescence system (Cytiva). ImageJ 1.47 (National Institutes of Health) was used for densitometry analysis.
Enhanced chemiluminescence system
Manufactured by Cytiva
Sourced in United States, United Kingdom, Japan, Germany, China, Sweden, Switzerland, Italy
The Enhanced Chemiluminescence System is a laboratory instrument designed to detect and quantify specific proteins or molecules in a sample. The system utilizes chemiluminescent detection, a technique that generates light through a chemical reaction, to provide sensitive and accurate results.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (18 mentions)
Nitrocellulose membrane, by Cytiva (12 mentions)
β actin, by Merck Group (11 mentions)
Polyvinylidene difluoride membrane, by Merck Group (10 mentions)
Bradford assay, by Bio-Rad (8 mentions)
Gapdh, by AbFrontier (8 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (7 mentions)
Pvdf membrane, by Cytiva (6 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (6 mentions)
Nitrocellulose membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (5 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cytiva (5 mentions)
Gapdh, by Abcam (4 mentions)
306 protocols using enhanced chemiluminescence system
1
Quantitative Analysis of Cartilage Proteins
2
Nuclear Protein Extraction and Immunoblotting
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Protein was extracted from cells using 1× RIPA buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors. Nuclear sub-fractionation was performed according to the previous protocol [19 (link)]. Proteins were quantified and resolved on an SDS-PAGE gel, transferred onto a PVDF membrane (Millipore), and immunoblotted with primary antibody, followed by incubation with secondary antibody. Antibody signal was detected using an enhanced chemiluminescence system (Cytiva). Antibodies used are listed in Supplemental Table 2 . For measurement of secretary HMGB1, culture medium was collected and spun down at 2000 × g for 5 min to remove any cell debris. Collected medium was denatured with SDS denaturing buffer. Western blotting was performed as described above.
3
Western Blot Protein Detection Protocol
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The protein was extracted and its concentration was determined by using a BCA protein concentration determination kit. The whole-cell lysates were separated by SDS-PAGE. Extracted protein were resolved by SDS-PAGE electrophoresis, and transferred to nitrocellulose filter membrane (NC). The membrane was blocked with Tris-buffered saline containing 0.1% Tween-20 (1×TBST) and 5% skimmed milk by incubating at room temperature for 60 min and washed with 1×TBST. The membrane was incubated with primary antibody (1:1000) dissolved in 1×PBS containing 5% BSA overnight at 4°C. After the incubation, the membrane was washed with 1×TBST and then incubated with secondary antibodies (1:5000) diluted in 1×TBST containing 5% skimmed milk. The membrane was further incubated at room temperature for 1 h, and washed with 1×TBST for three times. The target protein bands were visualized and recorded with a chemiluminescence imaging system (Tanon) using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech).
4
Immunoblot Analysis of Macrophage Markers
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Lung tissues or macrophages were collected and immediately homogenized in solubilizing buffer at 4°C (1% Triton X-100,100 mM Tris-HCl, pH 7.4, 100 mM sodium pyrophosphate,100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate, 2.0 mM PMSF, and 0.1 mg aprotinin/mL). Insoluble material was removed by centrifugation for 20 minutes at 9000g at 4°C. The protein concentration of the supernatants was determined by the Bradford assay. The samples were subjected to SDS-PAGE in a Bio-Rad (Hercules, CA) miniature slab gel apparatus (Mini-Protean). For immunoblot experiments, 0.2 mg of protein extracts from each tissue were separated by SDS-PAGE, transferred to PVDF membranes, and blotted with anti- iNOS, Arg-1, IRF5, IRF4 and GAPDH (abcam, USA). Proteins were visualized by chemiluminescence with an ECL kit (Millipore Corp., Billerica, MA, USA) and an enhanced chemiluminescence system (Amersham Life Science, Arlington Heights, IL, USA). The relative protein expressions were normalized to GAPDH. Band intensities were quantitated by optical densitometry (Scion Image software, ScionCorp, Frederick, MD).
5
Quantification of Immune Markers by Western Blot
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The tissue was homogenized with lysis buffer, an equal amount of protein (50 μg) was analyzed by 12% SDS–polyacrylamide gel electrophoresis, and the blotted membrane was blocked with 5% BSA in TBST for 60 min at room temperature, anti-Foxp3 (1:1000, Abcam), anti-CD25 (1:1000, Affinity Biosciences), anti-IL-17A (1:1000, Affinity Biosciences), anti-L-10 (1:1000, bs-6761R, Bioss) and FN-γ (1:1000, Affinity Biosciences) antibody overnight at 4 °C. GAPDH (1:1000.Sigma) was used as a loading control. The HRP-conjugated anti-rabbit or anti-mouse IgG (1:2000, Cell Signaling) was used as a secondary antibody and the membrane was developed using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).
6
Protein Expression Analysis in Cells
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Extract total proteins from cells, and then the protein content was evaluated. The protein was separated by electrophoresis on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a Hybond-P polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in 5% nonfat milk and incubated with Notch-1, Hes-1, Jagged-1, E-cadherin, and vimentin antibodies (1:1,000, CST, Danvers, MA, USA) 4 °C for the night. Then, incubation with the horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, CST, Danvers, MA, USA) and leave them at room temperature for 1 hour. After washing with PBS, the bound primary antibody was visualized with the Enhanced Chemiluminescence System (Amersham, Piscataway, NJ, USA) and exposed to film. -The same approach was applied to GAPDH (Solarbio, San Diego, CA, USA) for loading control. The relative density was analyzed with the Image J software.
7
Immunoblotting of Cellular Signaling Proteins
8
Western Blot Analysis of Stress Proteins
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As described in our previous study[8 (link)], the whole cell lysate protein was mixed with SDS-PAGE sample buffer and boiled for five minutes. Prepared protein samples were separated by SDS-PAGE electrophoresis and electrotransferred onto polyvinylidene fluoride membranes. After blocking, the membranes were blotted with primary antibodies against PDX1, HSP27, HSP60, and HSP70 (Santa Cruz Biotechnology). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized using an enhanced chemiluminescence system (Amersham, Piscataway, NJ, United States).
9
Western Blot Analysis of Inflammatory Proteins
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Western Blot Analysis was performed as described previously16 (link). Briefly, cell lysates (20 μg/lane) were separated on 10% SDS polyacrylamide gel and then were transferred to a poly (vinylidene fluoride) membrane. NLRP3 (Cat No.TA336883), cIAP1 (sc-271419) and 2 (Cat No.TA800041), c-Rel (Cat No.TA324346) and p52 (sc-7386) protein was detected by a mouse monoclonal IgG (OriGene Co. Ltd, Beijing, China and Santa Cruz Biotechnology Inc., USA) and visualized by the enhanced chemiluminescence system (Amersham, Arlington Heights, IL). The density of the bands was quantitated using the NIH image software package. The intensity of gene expression was judged by the ratio of their expression in PFDA treated groups to their corresponding expression in DMSO groups, and a ratio of more than 1.0 was considered to be an indication of over-expression.
10
Western Blot Analysis of Protein Levels
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Analysis of protein levels was performed using western blotting. Cell lysates from different treatment groups were prepared as described previously [14 (link), 15 (link)]. Proteins (25–40 μg protein) were electrophoresed on premade 10% Trisglycine gels (Invitrogen) and then transferred onto nitrocellulose membranes. After blocking in freshly prepared PBS containing 3% non-fat dry milk at room temperature for 30 min, the membranes were incubated with antibodies against DNMT1, DNMT3a and DNMT3b, CDKN1A, CDC2, pCDC2, and GADPH at 4 °C overnight followed by an anti-rabbit peroxidase-conjugated secondary antibody at 1:1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized on X-ray film using an enhanced chemiluminescence system (Amersham Life Science, Piscataway, NJ). Equal protein loading was verified using anti-b actin antibody. Experiments were repeated three times, and thus three western blots were run in each experiment, and representative blot is shown in each case.
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