Dermal fibroblast cells were lysed in solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8), 1% NP40 and 2.5% SDS supplemented with protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Fisher). Protein samples were loaded to a 4–20% Mini-PROTEAN TGX stain-free gel (Bio-Rad), transferred to a PVDF membrane and incubated with respective primary antibodies. The antibodies and the dilutions used in this study were the following: anti-SMC2 (Novus Biologicals, NB100-373), 1:2000; anti-SMC4 (Proteintech, 24758-1-AP), anti-ß-Actin (Cell Signaling, #4970), 1:1000. After incubating the membrane with horseradish peroxidase-conjugated secondary antibodies, the proteins were visualized with enhanced chemiluminescence detection (WesternBright ECL HRP substrate, Advansta) method.
Westernbright ecl hrp substrate
Manufactured by Advansta
Sourced in United States, Germany, Canada
WesternBright ECL HRP substrate is a chemiluminescent substrate for the detection of horseradish peroxidase (HRP) in Western blot applications. It generates a luminescent signal upon reaction with HRP, which can be detected using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Nitrocellulose membrane, by Bio-Rad (6 mentions)
Image lab software, by Bio-Rad (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Westernbright sirius hrp substrate, by Advansta (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
C digit blot scanner, by LI COR (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp system, by Bio-Rad (3 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (3 mentions)
Fusion fx imaging system, by Vilber (3 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Sds page, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Immobilon pvdf membrane, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Scrambled sirna, by Horizon Discovery (3 mentions)
Cl xposure film, by Thermo Fisher Scientific (3 mentions)
115 protocols using westernbright ecl hrp substrate
1
Immunoblotting of Dermal Fibroblast Proteins
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with respective drugs for 16 h and treated cells were washed and lysed in Pierce IP lysis buffer. SDS–PAGE was performed with 10–20 µg of proteins loaded per sample. Proteins were then transferred onto PVDF membranes. Blotted membranes were blocked with 5% milk in tris-buffered saline/Tween20 (TBST) for one hour, followed by three times 10 min washes in TBST. Primary antibodies were diluted in 5% BSA in TBST and incubations were performed overnight at 4 °C. After further washes, respective secondary HRP-conjugated antibodies diluted in 5% milk were incubated for one hour at room temperature. Detection of proteins on PVDF was carried out using WesternBright ECL HRP substrate or WesternBright Sirius HRP substrate (Advansta, San Jose, USA) and imaged with ChemiDoc XRS + System (Bio-Rad, Munich, Germany). Membranes were subjected to 10 min incubation with Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific, Waltham, USA) followed by TBST washes. After brief re-activation with methanol, membranes were blocked, and further probing of proteins was carried out.
3
Western Blotting Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were processed with 6× Laemmli sample buffer, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred onto the polyvinylidene difluoride (PVDF) membranes and blocked with 5% milk in TBST (Sigma-Aldrich, St. Louis, MO, USA). Western blotting was performed with specific antibodies at 4 °C overnight: anti-CD11b antibody (ABclonal, Woburn, MA, USA) and anti-GAPDH antibody (GeneTex, Irvne, CA, USA) (Supplementary Table S1 ). Membranes were washed with TBST twice and incubated with an HRP-labeled antibody at room temperature for 1 h. After incubation with an HRP substrate (Western Bright ECL HRP Substrate, Advansta, San Jose, CA, USA), images were taken with an Amersham™ Imager 600 (GE Healthcare, Chicago, IL, USA).
4
Western Blot Analytical Procedure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell extracts were prepared by washing the cells in cold PBS, pH 7.2, followed by solubilizing them in hot SDS-PAGE sample buffer (35 (link)), sonicating three times for 5 s at 20oC, and boiling for 5 min. Alternatively, in experiments analyzing sphingolipids or studies aimed to quantify expression of selected proteins (load), 2× concentrated SDS-PAGE sample buffer was added to the aliquots of sonicated lysates. Protein extracts (25 μg) were size fractionated in 13% SDS-PAGE gels and electrophoretically transferred onto nitrocellulose membrane (0.45 μm; GE Healthcare Life Sciences) using a Bio-Rad Mini-PROTEAN apparatus. The membranes were blocked with 2% BSA-TNT solution (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 0.1% Tween) and analyzed by immunoblotting with protein-specific antibodies. Bound primary antibodies were detected with the corresponding HRP-conjugated secondary antibodies. HRP signal in loads and phosphorylation studies was detected with chemiluminescence reagent (36 (link)), and HRP signals in pull-down studies were developed with WesternBright ECL HRP substrate (Advansta) or SuperSignal™ West Femto (Thermo Fisher Scientific) and collected with Luminescent Image Analyzer LAS 3000 (FujiFilm). Aida Image Analyzer, version 5.0 software (Raytest) was used for signal quantification.
5
Quantifying Bcl-2 Expression in Nanoceria-Treated Cells
6
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates, or cell fractions were incubated in Laemmli buffer at 55 °C for 10 min under reducing conditions. A total of 30 μg of protein from cell lysates or one-third of the volume collected in cell fraction preparations was separated by SDS-PAGE in 4–20% gradient gels (Biorad) and proteins were then transferred to PVDF membranes. After blocking with 5% TBS-milk, membranes were probed with primary antibodies overnight at 4 °C. The following primary antibodies were used: mouse anti-ABCA1 (Abcam; 1:1000), rabbit anti-GAPDH antibody (Millipore; 1:10,000), mouse anti-actin (CP01, Millipore 1:10,000), anti-MEK, anti-ERp72, anti-V5 and anti-Na+/K+ ATPase (Cell Signaling; all 1:1000), and rabbit anti-OSBPL7 (Sigma; 1:1000). After washing, membranes were incubated with anti-rabbit or and anti-mouse IgG-HRP antibodies (Promega, 1:10,000). Signals were detected after incubation with Westernbright ECL HRP substrate (Advansta) and luminescence signals were captured with Azure C600 Gel Imaging workstation (Azure Biosystems Inc, USA) or X-ray films.
7
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells or frozen tissues were lysed in Laemmli buffer containing a proteinase inhibitor cocktail (Santa Cruz). After centrifugation, the supernatant was harvested, and the protein concentration was detected by the BCA protein detection kit (Pierce). For immunoblotting, 15-30 µg of protein was separated by SDS-PAGE and then transferred to the PVDF membrane. The membrane was then blocked with 5 % bovine serum albumin in Tris-buffered saline containing Tween-20 (TBST). The membrane was incubated with the primary antibody overnight at 4 °C, then washing the membrane with TBST before incubation with the secondary antibody. Target proteins were detected with corresponding 1st and 2nd antibodies and visualized using Western bright ECL HRP substrate (Advansta Inc.). The primary antibody against β-actin was used as a control, and the result was defined by the ratio of target protein/β-actin.
8
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used for protein detection: TRIM71 (R&D system, AF5104, 1:3,000), hnRNP Q/R (Sigma, R5653, 1:5,000), LIN28B (Cell Signaling, #4196, 1:5,000), HA (Covance, MMS101R, 1:5,000), β ACTIN (Protein Tech, 66009-1-Ig, 1:10,000), anti-sheep (Jackson ImmunoResearch, 713-035-003, 1:5,000), anti-mouse (Sigma, A9044, 1:10,000), and anti-rabbit (Jackson ImmunoResearch, 211-032-171, 1:5,000). WesternBright ECL HRP substrate (Advansta K-12045-D20) or WesternBright Sirius HRP substrate (for detecting TRIM71 only) (Advansta K-12045-D10) were used for signal detection by the BioSpectrum AC Imaging System (UVP). Images were analyzed and quantified by the ImageLab Software (BIO-RAD).
9
Western Blot Analysis of Cell Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in a RIPA lysis buffer (Beyotime, Haimen, China) to extract total protein. Protein was separated on 12% SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Skimmed milk was used for blocking PVDF membranes. Immunoblotting of the membranes was conducted by incubation with primary antibodies: P27, CyclinD1 (CST, Danvers, MA, USA) and GAPDH (Affinity Biosciences, Cincinnati, OH, USA). Immunoblotting was carried out on a shaking table at 4 °C overnight. After incubation with a second antibody (CST) for one hour at room temperature and washing with TBS-T, a Western Bright ECL HRP substrate (Advansta, Menlo Park, CA, USA) was added to the membrane and it was visualized by an automated chemiluminescence image analysis system (Tanon 5200, Shanghai, China). Densities of protein bands were measured using ImageJ software (NIH, Bethesda, USA).
10
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human BMSCs were harvested by Pierce RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA). The crude extract was homogenized with a Branson S-450D Sonifier (Emerson Industrial Automation, St. Louis, MO, USA), followed by centrifugation at 14 000Âg and 4 °C for 15 min to remove cell debris. A total of 50-70 lg of protein from each individual sample was subjected to SDS-PAGE with a 4-12% gradient gel. The resolved proteins were transferred to PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were incubated at room temperature for 1 h in 3% bovine serum albumin, and reacted overnight at 4 °C with primary antibodies diluted in TBST buffer (50 mM Tris, 150 mM NaCl and 0.05% Tween 20, pH 7.5). After washing with TBST buffer, the blots were reacted for 45 min with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in TBST at a ratio of 1:5000 (v:v). Chemiluminescent signals of target proteins were produced with WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA) and detected with C-DiGit Ò Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA). Quantification of Western blot signals was performed with LI-COR Image Studio 4.0 software (LI-COR Biotechnology, Lincoln, NE, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!