After DKC-E70 treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000×g for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
Ecl western blotting substrate
Manufactured by Cytiva
Sourced in United States
The ECL Western blotting substrate is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It generates a luminescent signal upon interaction with the target protein, which can be captured and quantified using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (16 mentions)
Ripa buffer, by Boston BioProducts (15 mentions)
Phosphatase inhibitor cocktail, by Merck Group (13 mentions)
Protease inhibitor cocktail, by Merck Group (13 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
C digit blot scanner, by LI COR (4 mentions)
Un scan it gel version 5, by Silk Scientific (4 mentions)
Phosphatase inhibitor, by Merck Group (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Cyclin d1, by Abcam (2 mentions)
Radioimmunoprecipitation assay buffer, by Boston BioProducts (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Azure c600 device, by Azure Biosystems (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (1 mentions)
26 protocols using ecl western blotting substrate
1
Protein Extraction and Western Blot Analysis
2
Western Blot Protein Analysis
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The cells were washed with 1× phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000× g for 10 min at 4 °C. After determining the protein concentration by a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), the proteins were separated via SDS-PAGE and transferred to a PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and the chemiluminescence was detected using an ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized on Polaroid film.
3
Protein Expression Analysis in Cultured Cells
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Cells were plated at a density of 2 × 106 cells/well in 6-well plate and grown to 80% confluence. After treatment, the cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000 × rpm for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
4
Western Blot Analysis of Protein Expression
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After NAR treatment, cells were washed with 1×phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and centrifuged at 15,000×g for 10 min at 4°C. After determining protein concentration by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), the proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4°C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
5
Monitoring Epitope-Tagged Protein Levels
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Bacteria producing epitope-tagged proteins were inoculated into N minimal medium (pH 7.4) with 10 mM MgCl2 (high Mg2+) for overnight growth at 37°C with aeration, and the cultures were shifted to N minimal medium (pH 7.4) with 10 μM MgCl2 (low Mg2+) and grown for 4 h. Bacterial cells were harvested and lysed by sonication. The protein concentration of whole-cell lysates was determined by using the BCA Protein Assay kit (Pierce). Protein samples were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes (Amersham), and the PhoP-HA, SlyA-FLAG, and EmrR-FLAG proteins were monitored with ECL Western blotting substrate (Amersham) after incubation with monoclonal anti-HA or anti-FLAG M2 antibody (Sigma-Aldrich).
6
Protein Extraction and Western Blot Analysis
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After PF treatment, cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000 × g for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5 % non-fat dry milk in Tris-buffered saline containing 0.05 % Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5 % non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
7
Protein Separation and Western Blot Analysis
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Samples were mixed with a 4× lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) containing a sample-reducing agent (Invitrogen) and boiled at 95 °C for 10 min. Proteins were separated on 4–12% Bis-Tris gels (Invitrogen). The gel was stained by Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules, CA, USA) for 1 h with gentle agitation. The stained gel was then treated with a destaining solution (Biosesang, Sungnam, Korea) and agitated until the protein bands were visible.
For Western blotting, the gel was transferred onto a polyvinylidene fluoride membrane (Invitrogen) using an iBlot Gel Transfer Device (Invitrogen). The membranes were blocked with 2% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) for 1 h at room temperature (RT) with shaking, washed thrice with PBS-T for 10 min, and then incubated overnight with a home-made primary antibody against FMDV VP1 at 4 °C. The following day, the membranes were washed thrice with PBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Invitrogen) for 1 h at RT. The antibody–antigen complexes were visualized with ECL Western blotting substrate (Amersham, Buckinghamshire, UK) using an Azure C600 device (Azure Biosystems, Dublin, CA, USA).
For Western blotting, the gel was transferred onto a polyvinylidene fluoride membrane (Invitrogen) using an iBlot Gel Transfer Device (Invitrogen). The membranes were blocked with 2% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T) for 1 h at room temperature (RT) with shaking, washed thrice with PBS-T for 10 min, and then incubated overnight with a home-made primary antibody against FMDV VP1 at 4 °C. The following day, the membranes were washed thrice with PBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Invitrogen) for 1 h at RT. The antibody–antigen complexes were visualized with ECL Western blotting substrate (Amersham, Buckinghamshire, UK) using an Azure C600 device (Azure Biosystems, Dublin, CA, USA).
8
Protein Expression Analysis in Cultured Cells
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Cells were plated at a density of 2 × 106 cells/well in 6-well plate and grown to 80% confluence. After treatment, the cells were washed with 1 × phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000 × rpm for 10 min at 4 °C. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). The proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4 °C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
9
Western Blot Analysis of Protein Expression
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Cells were washed twice with PBS and then lysed in RIPA lysis buffer (Sigma #R0278) (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, Proteinase Inhibitor 1x). Post sonication, cell lysates were centrifuged at 14000 g for 10 min at 4 °C, and the supernatants were used for Western blotting. The lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and then stained with 0.1% Ponceau S solution (Sigma #P3504) to ensure equal loading of the samples. After being blocked with 5% non-fat milk for 60 min, the membranes were incubated with primary antibodies ABCG2 (Cell Signaling #42078); c-Myc (Cell Signaling #5605); Cyclin D1 (Abcam #ab134175); Bcl-xL (Cell Signaling #2764); Bax (Cell Signaling #5023); β-Actin (Cell Signaling #4970) overnight, and the bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies using the ECL Western Blotting Substrate (Amersham, GE #RPN2106) on a Chemi-Doc (Bio-Rad) imaging analyzer. The Primary antibodies concentrations are described in Supplementary Table 3 .
10
Western Blot Analysis of Kahweol Effects
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After kahweol treatment, cells were washed with 1×phosphate-buffered saline (PBS), and lysed in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) supplemented with protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Sigma-Aldrich), and centrifuged at 15,000×g for 10 min at 4°C. After determining protein concentration by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA), the proteins were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked for non-specific binding with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then incubated with specific primary antibodies in 5% non-fat dry milk at 4°C overnight. After three washes with TBS-T, the blots were incubated with horse radish peroxidase (HRP)-conjugated immunoglobulin G (IgG) for 1 h at room temperature and chemiluminescence was detected with ECL Western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) and visualized in Polaroid film.
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