Cells transfected for 48 h were harvested from all groups, followed by lysis using RIPA lysis buffer (Beyotime, Shanghai, China). The cells were then centrifuged for 15 min at 12,000 g and 4°C to collect protein supernatants, and the BCA kit (Thermo Fisher Scientific, Inc.) was used to measure protein content. Later, proteins were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) as well as 2 h of blocking using 5% nonfat milk at ambient temperature. The primary antibodies (Abcam, Shanghai, China), anti-HMGB2 (ab124670, 1: 10,000), and anti-GAPDH (ab9485, 1:2500) were added, followed by an overnight incubation at 4°C. The next day, the membrane was rinsed with Tris-buffered saline with Tween-20 (TBST) and incubated with horseradish peroxidase (HRP)-labeled secondary antibody (ab205718, 1:5000) for 2 h at 37°C. After the PVDF membranes were washed with TBST three times, bands were visualized using ECL chemiluminescence (Beyotime, Shanghai, China) [40 (link)].
Ecl chemiluminescence
Manufactured by Beyotime
Sourced in China
ECL chemiluminescence is a laboratory technique that utilizes a luminescent chemical reaction to detect and quantify specific proteins or molecules in a sample. The core function of this equipment is to generate a luminescent signal that can be measured and analyzed to provide information about the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Pvdf membrane, by Roche (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab9485, by Abcam (2 mentions)
Cleaved caspased 3, by Wanlei (2 mentions)
Bcl 2, by Wanlei (2 mentions)
Bcl2 associated x (bax), by Wanlei (2 mentions)
Ab124670, by Abcam (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Anti p62, by Merck Group (1 mentions)
Ripa lysis kit, by Beyotime (1 mentions)
Anti lc3, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Quantity one software version 4, by Bio-Rad (1 mentions)
Skim milk powder, by Beyotime (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Yeasen (1 mentions)
14 protocols using ecl chemiluminescence
1
Western Blot Protein Analysis Protocol
2
Quantifying Autophagy Markers in EPCs
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We extracted protein from EPCs with the RIPA Lysis Kit (Beyotime). The total protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). All of the samples, each containing 30 g protein, were boiled with loading buffer for 5 min and then loaded onto a 12% SDS-PAGE gel for 1.5 h and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk in TBST for 1 h and then incubated with primary antibodies against four autophagy markers, anti-P62 (Sigma), anti-LC3 (Sigma), phospho-p70 S6 Kinase, Beclin-1 and anti-actin, overnight. Next, we removed the excess antibodies by washing with TBST and incubated the membranes with horseradish peroxidase-coupled secondary antibodies for 1 h. The immunoblots were visualized using ECL chemiluminescence (Beyotime) and quantified with Quantity One software (Bio-Rad, USA).
3
Liver Protein Quantification and Expression
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Liver tissues were homogenized in RIPA buffer. Protein concentration wass measured by the BCA method (KeyGENbio, Nanjing, China). Protein samples were subjected to 12% SDS-polyacrylamide gels and then electrotransferred onto a PVDF membrane. After incubation with β-actin, Bax, Bcl-2, Caspase-3, Caspase-9, TNF-α, NF-κB, AMPK-α, PPAR-α, SREBP-1c primary, and secondary goat anti-mouse antibodies, proteins were visualized using ECL chemiluminescence (Beyotime, Shanghai, China). WB bands and quantitation were analyzed using Quantity One software (version 4.6.2) (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Evaluating Genotoxicity of Nano-Conjugated Gefitinib
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A549 and H1299 cells were cultured in six-well plates with a seeding density of 1 × 106 cells/well for 24 h. Subsequently, the cells were treated with 50 μM of GEF, GEF@NCA, or GEF@TSBO for an additional 24 h. After treatment, the cells were harvested and lysed using protease inhibitors on ice for 30 min. The lysate was then centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was quantified with a BCA Protein Assay Kit (Yeasen Bio., Shanghai, China). Proteins were separated on a 10% Bis-Tris polyacrylamide gel (Beyotime, Nantong, China) and then transferred onto a PVDF membrane (BioRad, Hercules, CA, USA). The membrane was blocked with 5% skim milk powder (Beyotime, Nantong, China) and incubated with primary antibodies against GAPDH (Servicebio, Wuhan, China), γ-H2AX (CST, Danvers, MA, USA), and PARP (CST, USA) overnight. After an hour of incubation, protein expression levels were detected using ECL chemiluminescence (Beyotime, Nantong, China) with a suitable secondary antibody (Yeasen Biotechnology, Shanghai, China).
5
Protein Expression Analysis in Skin Tissues
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Protein samples were obtained from the skin tissues and melanocytes according to the Protein Extraction Kit (Sangon Biotech., China). Protein content was quantified using the Bio-Rad protein assay. Then, samples were diluted, heated for denaturation, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA) and blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% non-fat dry milk for 1 h. Then, 1:1,000 dilutions of rabbit polyclonal primary antibody anti-mc1r-C (HuaAn Biotechnology Co., Ltd., China) or anti-β-actin (loading control) were incubated at 4°C overnight. The following day, the membrane was rinsed in TBST three times and incubated with a fluorescent secondary anti-rabbit antibody (1:5,000) for 1 h at 37°C. After washing three times with Tris-buffered saline at 5 min per wash, the proteins were detected using ECL chemiluminescence (Beyotime, China) by image software (Bio-Rad Laboratories, PA, USA).
6
Protein Expression Analysis in Cell Signaling
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According to the manufacturer's protocol [19 (link)], proteins were extracted using RIPA Lysis Buffer (Beyotime, China) and protein concentration was measured using a bicinchoninic acid assay (Beyotime, China). Subsequently, the prepared protein was separated by 12% polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, Switzerland). After blocked with skim milk, the PVDF membranes were subjected to incubation with primary antibodies: Bcl-2, Bax, cleaved-Caspased-3, GCR, SIRT1, PGC-1α, and BDNF (1 : 500, Wanleibio Wuhan, China) overnight at 4°C. On the following day, the membranes were incubated with the secondary antibody at 37°C for 45 min and the intensity of protein expression was detected by ECL chemiluminescence (Beyotime, Beijing, China). Protein expression levels were semiquantified using ImageJ software (version 1.8.0; National Institutes of Health).
7
Protein Expression Analysis in Corneal Tissues
8
Protein Extraction and Immunoblotting
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DEFs were lysed in radio immunoprecipitation assay buffer supplemented with protease inhibitors (Sigma-Aldrich) on ice for 30 min. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). Immunoblotting was performed as described previously (19 (link)). Immunoreactive bands were visualized and analyzed using commercial enhanced ECL chemiluminescence (Beyotime, China).
9
Western Blot Analysis of Cellular Signaling
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A lysis buffer (RIPA, Beyotime, China) was added to each group of cells or tissues to fully lyse and extract total protein from the cells. Lysates were electrophoresed on 6-12% gels using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Protein isolates were transferred to PVDF membranes (Millipore, USA) and blocked in 5% skim milk powder solution for 1 h at room temperature. The cells were incubated with primary antibody overnight at 4°C. Primary antibodies against GPX4 (Proteintech, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Cleaved Caspase-3 (CST, USA), p-ERK (CST, USA), ERK (CST, USA), p-p38 (CST, USA), p38 (CST, USA), p-JNK (CST, USA), JNK (CST, USA), and β-actin (Santa Cruz, USA) were used. The appropriate secondary antibody (1 : 5000; Cell Signaling Technology, USA) was incubated for 1 hour at 37°C on a shaker, and the bands were observed using ECL chemiluminescence (Beyotime Biotechnology, China). ImageLab software was used for data analysis.
10
Western Blot Analysis of Autophagy and EMT Markers
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Proteins of lung tissues and RLE‐6TN cells were extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China). After quantification, the protein samples were subjected to SDS-PAGE, then blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies LC3B (ab48394, 1 µg/ml, Abcam), Beclin-1 (ab207612, 1:2000, Abcam), p62 (ab109012, 1:10000, Abcam), E-cadherin (ab231303, 1 µg/ml, Abcam), N-cadherin (ab76011, 1:5000, Abcam), Vimentin (ab92547, 1:1000, Abcam), α-SMA (ab124964, 1:10000, Abcam), collagen I (ab270993, 1:1000, Abcam), Fibronectin 1 (ab45688, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), PI3K (ab191606, 1:1000, Abcam), p-Akt (ab38449, 1:500, Abcam), Akt (ab38449, 1:500, Abcam), p-mTOR (AP0094, 1:500, ABclonal), mTOR (A2445, 1:500, ABclonal), CDC27 (A3333, 1:1000, ABclonal), and GAPDH (ab8245, 1:2000, Abcam) at 4°C overnight, followed by incubation with secondary antibody (bs-0295 G-HRP, 1:1000, Bioss). ECL chemiluminescence (Beyotime) was applied for analysis.
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