For western blotting, total cells were washed with ice-cold PBS, lysed in SDS buffer on ice and boiled for 10 min at 100°C. Then, proteins in the samples were separated by SDS–PAGE, transferred onto PVDF membranes (Millipore) and probed with the corresponding primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. An ECL Kit (MultiSciences, Hangzhou, Zhejiang, China) was used to visualize the bands.
Ecl kit
Manufactured by MultiSciences Biotech
Sourced in China
The ECL kit is a laboratory equipment used for the detection and quantification of proteins through Western blotting. It contains reagents that facilitate the chemiluminescent reaction, enabling the visualization of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ab9485, by Abcam (3 mentions)
Pvdf membrane, by Wuhan Servicebio Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Wuhan Servicebio Technology (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Protease inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
Ripa lysis buffer, by MultiSciences Biotech (1 mentions)
Bca protein assay kit, by MultiSciences Biotech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Ab1791, by Abcam (1 mentions)
Ab108341, by Abcam (1 mentions)
Ab18203, by Abcam (1 mentions)
Bs 12147r, by Bioss Antibodies (1 mentions)
Ab8227, by Abcam (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ab1872, by Abcam (1 mentions)
10 protocols using ecl kit
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Lung Tissue
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Lung tissues were homogenized in RIPA lysis buffer (MultiSciences, Hangzhou, China) containing protease inhibitor cocktail (Servicebio, Wuhan, China). The BCA protein assay kit (MultiSciences, Hangzhou, China) was used for protein quantification. Samples were separated by 10% SDS-PAGE and then electro-transferred onto polyvinylidene difluoride membranes. After blocking for 1 h with 5% nonfat dry milk at room temperature, blots were incubated at 4°C with the following specific antibodies: anti-p-PI3K (4228S), anti-PI3K (4292S), anti-p-AKT (4060S), anti-AKT (9272S), anti-p-mTOR (2971S), anti-mTOR (2972S), anti-caspase-3 (9662S), anti-cleaved caspase-3 (9664S), anti-p-IκBα (2859S), anti-IκBα (4812S), anti-p-p65 (3033S), anti-p65 (8242S), and anti-HMGB1 (6893S) (all from Cell Signaling Technology); anti-BAX (ab32503, Abcam) and anti-BCL-2 (ab182858, Abcam); and anti-GAPDH (GB11002, Servicebio). After incubating with primary antibody overnight, and washing three times with tris-buffered saline containing Tween 20, blots were incubated with the corresponding HRP-conjugated secondary antibody for 1 h at room temperature. Immunoreactivity was determined using an enhanced chemiluminescence reagent ECL kit (MultiSciences, Hangzhou, China).
3
Western Blot Analysis of EMT Markers
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Total proteins were extracted with RIPA lysis buffer (Beyotime, China) and protein concentrations quantified by a BCA assay. Equal amounts of protein were separated by 8–12% SDS-PAGE (Servicebio, China) and transferred onto PVDF membranes (Servicebio, China). After blocking with 5% nonfat dry milk, the membranes were incubated with the following primary antibodies: anti-N-cadherin (ab18203, Abcam, UK), anti-E-cadherin (3195, Cell Signaling Technology, CST, USA), anti-Snail1 (3879, CST), anti-TWIST1 (25465-1-AP, Proteintech, USA), anti-ZIC5 (ARP33669-P050, Aviva Systems Biology, USA; bs-12147R, Bioss, China), anti-β-actin (ab8227, Abcam), anti-β-catenin (51067-2-AP, Proteintech), anti-TCF4 (22337-1-AP, Proteintech), anti-Histone H3 (ab1791, Abcam), anti-Tubulin (2148, CST), anti-AR-V7 (19672, CST), anti-AR (ab108341, Abcam), and anti-GAPDH (ab9485, Abcam). After incubation with suitable HRP-conjugated antibodies, the blots were visualized using an ECL kit (Multisciences, China).
4
Western Blot Analysis of Inflammatory Markers
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Cells and tissues were collected and lysed with RIPA buffer containing phenylmethanesulfonylfluoride (Beyotime, China). Protein concentration was determined with a BCA assay kit (Beyotime, China). Equivalent amounts of total protein were separated by 10% sodium dodecyl sulfate polyacrylamide (servicebio, China) gel electrophoresis and then transferred to polyvinylidene fluoride membranes (servicebio, China). Blocking was performed in TBST containing 5% nonfat milk, and membranes were incubated with primary antibody overnight at 4 °C. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. An ECL kit (Multisciences, China) was used to detect the proteins. Image J software (NIH, MD, USA) was used to evaluate the relative expression levels of different proteins. Primary antibodies for BRD4 (ab128874), caspase-1 (ab1872), Il-1β (ab7632), cleaved N-terminal GSDMD (ab215203), vimentin (ab92547), caspase-3 (ab2302) and GAPDH (ab9485) were purchased from Abcam. Antibodies for Cl-caspase-1 (4199), E-ca (3195), NLRP3 (15101), NF-κB-p65 (8242), phospho-NF-κB-p65 (3033), IkBa (4812), and p-IkBa (2859) were from Cell Signaling Technology. Goat and anti-rabbit secondary antibodies were purchased from Wuhan Boster Bio-engineering Limited Company, China.
5
Protein Expression Analysis by Western Blot
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Cells and tissues were collected and lysed with RIPA buffer containing phenylmethanesulfonylfluoride (Beyotime, China). Equivalent amounts of total protein were separated by 10% SDS-PAGE (Servicebio, China), then transferred to PVDF membranes (Servicebio, China). The membranes were then blocked in TBST containing 5% nonfat milk, and the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with secondary antibodies. An ECL kit (Multisciences, China) was used to detect proteins. Primary antibodies against FOXO1 (2880) and cyclin D1 (2978S) were purchased from Cell Signaling Technology. Antibodies against p21 (ab109520) and GAPDH (ab9485) were purchased from Abcam.
6
Western Blot Analysis of RBC-Derived Extracellular Vesicles
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A total of 10 μg of RBC‐EV, cell or tissue lysates was resuspended in 5 × SDS loading buffer, subsequently incubated at 100°C for 5 min and centrifuged at 12,000 × g for 10 min. Then, the supernatants were separated by 10% SDS‐polyacrylamide gel (Thermo Fisher Scientific) electrophoresis and transferred to PVDF membranes (Millipore), which were blocked with 5% skim powdered milk (w/v) for 1.5 h, incubated with the relevant primary antibodies at 4°C overnight, and then incubated with secondary antibodies at RT for 2 h. An ECL Kit (MultiSciences, Hangzhou, Zhejiang, China) was used to detect the bands. The antibodies used and the corresponding dilutions are listed in Table S2 .
7
Western Blotting of Cells and EVs
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For western blotting, total cells and EVs were washed with ice-cold PBS, lysed in SDS buffer on ice and boiled for 10 min at 100 °C. Then, proteins in the samples were separated by SDS-PAGE, transferred onto PVDF membranes (Millipore) and probed with the corresponding primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. An ECL Kit (MultiSciences, Hangzhou, Zhejiang, China) was used to visualize the bands.
8
Western Blot Analysis of Key Signaling Pathways
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The experimental steps of Western blot were conducted by our previous literature.21 (link) The primary antibodies listed below were incubated with the membranes at 4 °C and stayed overnight (at a 1:1,000 dilution): α-SMA, transforming growth factor (TGF)-β1, collagen-I, collagen-III, MMP-2, MMP-9, NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, and IL-18 (ABclonal Technology, Wuhan, China); ODC, SSAT, p-Smad-2, t-Smad-2, p-Smad-3, t-Smad-3, and Smad-7 (Cell Signaling Technology, Danvers, Massachusetts); and ubiquitin, β-actin, and β-tubulin (Santa Cruz Biotechnology). The membranes were incubated for 1 h at room temperature with secondary antibodies (diluted at 1:10,000, Proteintech, Wuhan, China). The specific complexes treated with an ECL kit (MultiSciences, Hangzhou, China) were detected using a multiplex fluorescent imaging system.
9
Western Blot Analysis of EVs, Cells, and Tissues
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Total protein was extracted using RIPA buffer (Beyotime, Shanghai, China) or cell lysis buffer (CST, USA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail. Protein concentrations were determined by the BCA protein assay kit. A total of 10 µg of the indicated EVs, cell lysates or tissue lysates were resuspended in 5 × SDS loading buffer, subsequently incubated at 100°C for 10 min. Then, the supernatant was separated by 10% SDS‐polyacrylamide gel (Thermo Fisher Scientific) electrophoresis and transferred to PVDF membranes (Millipore), which were blocked with 5% non‐fat powdered milk in PBS with Tween 20 buffer (PBST) for 1 h, incubated with the relevant primary antibodies at 4°C overnight, and then incubated with secondary antibodies at RT for 1 h. An ECL kit (MultiSciences, Hangzhou, Zhejiang, China) was used to detect the bands. The antibodies used and the corresponding dilutions are listed in Table S2 .
10
Western Blot Analysis of NLRP3 and Caspase-1
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Cell or tissue lysate was resuspended in 5× SDS loading buffer, subsequently incubated at 100°C for 5 min and centrifuged at 12,000 × g for 10 min. Protein concentrations were detected using a BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20 μg of protein from the tissue or cell lysate was separated by SDS-PAGE gel (Thermo Fisher Scientific) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was blocked using 5% nonfat milk for 2 h at room temperature and then incubated with appropriate primary antibodies: anti-NLRP3 (Abclonal, Wuhan, China) (1 : 1,000) and anti-Caspase-1 (Abclonal, Wuhan, China) (1 : 1,000) in blocking buffer overnight at 4°C. Anti-β-actin (HuaBio, Shanghai, China) (1 : 2,000) was used as a loading control. After washing three times with PBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1.5 h at room temperature. The bands were detected using an ECL kit (MultiSciences, Hangzhou, Zhejiang, China).
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