Lung tissue protein was extracted by RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd., China). After the quantification of proteins, they were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on NC membranes. The membranes were blocked with 5% skim milk and then incubated with primary antibodies against the target proteins (Abcam, Cambridge, UK; Abmart Inc., Shanghai, China) at 4°C overnight. Then, the membranes were incubated with secondary antibodies (Beijing Emarbio Science & Technology Co., Ltd.; China; anti-rabbit, Thermo Fisher Scientific, Inc., USA) for 1 h. The membranes were treated with enhanced chemiluminescence detection reagent (Biosharp, Shanghai, China), and the protein bands were visualized using Biorad Chemidoc XRS+ ChemiLuminescence Imaging System (Bio-Rad, USA). The relative band density was determined by Image J (National Institutes of Health, Bethesda, MD, USA).
Biorad chemidoc xrs chemiluminescence imaging system
Manufactured by Bio-Rad
Sourced in United States
The Biorad Chemidoc XRS+ ChemiLuminescence Imaging System is a versatile laboratory equipment designed for the detection and analysis of chemiluminescent signals. It is capable of capturing high-quality images of chemiluminescent samples, such as Western blots, for further analysis and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection reagent, by Biosharp (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Rapidstep ecl reagent, by Merck Group (1 mentions)
Toll like receptor 4 tlr4, by Abcam (1 mentions)
P ikbα, by Abcam (1 mentions)
Bcl 2 associated x protein bax, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Beyoecl plus kit, by Beyotime (1 mentions)
5 protocols using biorad chemidoc xrs chemiluminescence imaging system
1
Western Blot Analysis of Lung Proteins
2
Western Blot Analysis of Bim and GAPDH Proteins
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Western blot analysis was performed as described previously (Wang et al., 2020 (link)). Briefly, cells (CNE-2, H1299, and HSF) were harvested, washed and lysed with 1 × RAPI buffer. Protein concentration was determined by the Thermo BCA protein assay Kit. Equal amounts of protein from cell lysates were solubilized in 5 × SDS sample buffer and separated on 8–10% SDS polyacrylamide gels, and transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat milk in TBST and incubated with primary antibodies against Bim and GAPDH proteins at 4°C overnight. Afterwards, the membranes were washed and incubated with a secondary antibody against rabbit IgG for 1 h, followed by washing and transferring into ECL solution (Millipore, Darmstadt, Germany), and scanned under the Bio-Rad ChemiDoc XRS + Chemiluminescence imaging system (Bio-Rad, Hercules, CA, United States). The results were measured by ImageJ software.
3
Western Blot Characterization of C28/I2 Cells
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After the treated C28/I2 cells were harvested, 30 μg of protein was extracted by RIPA lysis buffer (Beyotime, Shanghai, China), separated on SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Subsequently, the membranes were immersed in 5% nonfat milk for 2 h and incubated with primary antibodies against B cell lymphoma-2 (Bcl-2, 1:1,000, Abcam, Cambridge, MA, USA), Bcl-2-associated X protein (Bax, 1:2,000, Abcam), SOX11 (1:2,000, Abcam), phosphorylation-P65 (p-P65, 1:1,000, Thermo Fisher Scientific), P65 (5 µg/mL, Thermo Fisher Scientific), IkB-α (1:2,000, Abcam), p-IkB-α (1:3,000, Abcam), Toll-like receptor 4 (TLR4, 1:1,000, Abcam), or GAPDH (1:3,000, Abcam) at 4°C overnight. The membranes were then probed with a secondary antibody conjugated with horseradish peroxidase (1:5,000, Abcam) for 1 h at 37°C. The blots were visualized by RapidStep ECL Reagent (Millipore Corp., Billerica, MA, United States), and the results were observed using Bio-Rad ChemiDoc XRS + Chemiluminescence Imaging System.
4
SDS-PAGE and Western Blot Analysis
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Proteins were extracted by 2x protein loading buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 5% 2-hydroxy-1-ethanethiol, 20% glycerol, 0.01% bromphenol blue). Cells (2 × 106) were added with 100 µl loading buffer, boiled at 100°C for 5 minutes, then frozen for 5 minutes, repeated 3 times. Proteins from cell lysates (10 µl) were electrotransferred to nitrocellulose membranes after separated on 10% SDS-PAGE. Before being blotted with primary antibody overnight at 4°C, membranes were sealed for 1 h at room temperature in Tris-buffered saline-0.05% Tween-20 (TBST) containing 5% nonfat dry milk. After 3 × 10 min washes in PBS, membranes were incubated with peroxidase-conjugated secondary antibody for 1 hr. Following 3 additional 10- min washes with TBST, the proteins were imaged by enhanced chemiluminescence detection reagent and detected with Bio-Rad ChemiDoc XRS+ chemiluminescence imaging system (Bio-rad laboratories Inc.).
5
Quantification of p120ctn and Kaiso in H1650 cells
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All proteins in the H1650 cell culture supernatant were analyzed directly for p120ctn and Kaiso and prepared in ice-cold RIPA lysis buffer for 30 min, followed by centrifugation at 12 000 rpm for 30 min at 4°C. Total protein concentrations were determined by BCA assay (Beyotime, Jiangsu, China), loaded onto SDS-PAGE gels for electrophoresis at a constant voltage, and electrotransferred onto PVDF membranes (Millipore, USA) for Western blot analysis, which were blocked with 5% BSA. Afterwards, the membranes were probed with the primary antibody (BD Transduction Laboratories, Santa Cruz Biotechnology, INC) at 4°C overnight, followed by washing 3 times with TBS-T buffer and incubated with secondary antibody (Santa Cruz Biotechnology, USA) at room temperature for 1 h. Then, the membranes were washed 3 times, visualized with an BeyoECL Plus kit (Beyotime Biotechnology, China), exposed, and processed with the Bio-Rad ChemiDoc XRS+ Chemiluminescence imaging system (Bio-Rad, USA), with GAPDH as a control for each sample. All experiments were repeated 3 times.
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