Cell lysates were prepared with RIPA buffer (Thermo Scientific). Immunoreactive bands were detected by using the Immobilon ECL substrate kit (Millipore, Merck KGaA, Germany). Antibodies used included primary antibodies against TFRC (Cat. No: ab218544, 1:1000 dilution, Abcam, USA), E-cadherin (Cat. No: ab1416, 1:1000 dilution, Abcam, USA) and β-actin (Cat. No: ab8226 1:1000 dilution, Abcam,); HRP-conjugated secondary goat anti-mouse (Cat. No: SA00001–1) or goat anti-rabbit (Cat. No: SA00001–2) antibodies (1:4000 dilution, Proteintech, USA).
Immobilon ecl substrate kit
Manufactured by Merck Group
Sourced in Germany, United States
The Immobilon ECL substrate kit is a laboratory product designed for the detection of proteins in Western blot analysis. It provides the necessary reagents to generate a chemiluminescent signal, which can be captured and quantified using imaging equipment. The kit allows for the sensitive and specific detection of target proteins immobilized on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Biospectrum 600 imaging system, by Analytik Jena (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Sds page, by Beyotime (2 mentions)
Complete protease inhibitor mixture, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab1416, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab75234, by Abcam (1 mentions)
Ab11350, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32072, by Abcam (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho pi3 kinase p85, by Cell Signaling Technology (1 mentions)
14 protocols using immobilon ecl substrate kit
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blotting
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The total protein of samples was extracted using RIPA lysis buffer (Beyotime). Identical quantities of proteins were separated by SDS/PAGE gel, and transferred to PVDF membranes (Millipore, Bedford, MA, U.S.A.). The membranes were blocked with 5% non-fat milk for 2 h, and probed with primary antibodies at 4°C overnight. Afterward, PVDF membranes were incubated with the specific HRP–conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by using the Immobilon ECL substrate kit (Millipore), and quantitated by ImageJ 3.0 software (http://imagej.nih.gov/ij/ ). GAPDH was used as the internal reference.
3
Western Blot Analysis of Wnt/β-catenin Pathway
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Total protein was extracted, separated, and transferred to polyvinylidene difluoride (PVDF, Millipore, Molsheim, France) membranes, as described previously.15 (link) After being blocked with 5% non-fat milk, the membranes were probed with primary antibodies overnight at 4 °C, followed by the incubation with horseradish peroxidase (HRP)-conjugated IgG (ab6721, Abcam, Cambridge, UK; dilution 1 : 10 000) as the secondary antibody. Immunoreactive bands were detected using the Immobilon ECL Substrate Kit (Millipore) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Primary antibodies were used as follows: anti-FZD5 (ab75234, Abcam; dilution 1 : 800), anti-β-catenin (ab32572, Abcam; dilution 1 : 5000), anti-phosphorylated (p)-β-catenin (ab11350, Abcam; dilution 1 : 1000), anti-c-Myc (ab32072, Abcam; dilution 1 : 1000), anti-Cyclin D1 (ab16663, Abcam; dilution 1 : 100) and anti-GAPDH (ab181602, Abcam; dilution 1 : 10 000).
4
Western Blot Analysis of PI3K/AKT/mTOR Pathway
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (CWBIO, China) with protease and phosphatase inhibitors (CWBIO, China). An equal amount of total protein lysates (30 ng) was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane and then incubated with primary antibodies specific for AGO1 (1:1,000 dilution; Proteintech, USA), anti-PI3Kinase p85 (1:1,000 dilution; Cell Signaling Technology, USA), phospho-PI3Kinase p85 (1:1,000 dilution; Cell Signaling Technology, USA), anti-AKT kinase (1:1,000 dilution; Cell Signaling Technology, USA), phospho-AKT (Ser 473, 1:1,000 dilution; Cell Signaling Technology, USA), anti-mTOR (1:1,000 dilution; Cell Signaling Technology, USA), phospho-mTOR (1:1,000 dilution; Cell Signaling Technology, USA), and GAPDH (1:1,000 dilution; Proteintech, China) at 4°C overnight, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. Bands were visualized using Immobilon ECL substrate kit (Millipore, Germany), and the images were captured via Bio Spectrum 600 Imaging System (UVP, USA).
5
Western Blot Analysis of Protein Expression
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Total proteins were extracted using radio immunoprecipitation (RIPA) lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 50 mM Tris-HCl, 0.1% SDS, 150 mM NaCl, pH 7.5) containing 1% protease inhibitor. The protein sample (30 µg in each group) was separated by 10% SDS-PAGE and transferred onto the nitrocellulose membranes (Millipore, Bedford, MA, USA). After being blocked with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with anti-GAPDH (Abcam, Cambridge, UK) (ab37168, 1:3000), anti-Vimentin (Abcam, Cambridge, UK) (ab8978, 1:3000), anti-E-cadherin (Abcam, Cambridge, UK) (ab40772, 1:3000), and anti-EN2 (ab45867, 1:500) primary antibodies (Abcam, Cambridge, UK) at 4°C for 12 h. Then, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at 4°C for 2 h. Finally, the intensity of bands was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc.) with Immobilon ECL substrate kit (Millipore, Billerica, MA, USA). GAPDH was utilized as an internal control.
6
Western Blot Protein Analysis
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Cell lysates were prepared with RIPA buffer (Beyotime). Identical quantity of protein samples were separated by SDS-polyacrylamide gels, and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Following blocking in 5% nonfat milk for 1 h, the membranes were incubated with specific primary antibodies and HRP-conjugated secondary antibody. The protein bands were visualized by the Immobilon ECL substrate kit (Millipore), and β-actin was employed as an internal control.
7
Western Blot Analysis of Key Proteins
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Western blot was performed using previously described protocols. Briefly, equal amount of total protein (20–40 μg) was separated by SDS-PAGE (Beyotime Biotechnology, Shanghai, China), transferred into the PVDF membrane (Millipore, USA), and then incubated overnight with primary antibodies specific for cyclin D1 (#A11022, ABclonal, China), VEGFR2 (#A5609, ABclonal, China), fibronectin (#A12932, ABclonal, China), α-SMA (#A7248, ABclonal, China), VEGFA (#ab52917, Abcam), cyclin A2 (#ab181591, Abcam), PDGFR (#ab69506, Abcam), collagen I (#ab138492, Abcam), collagen III (#ab184993, Abcam), elastin (#ab213720, Abcam), GAPDH (#10494-1-AP, Proteintech, China), ERK (#4695, Cell Signaling Technology), p-ERK (#4370, Cell Signaling Technology), AKT (#4691, Cell Signaling Technology), and p-AKT (#4060, Cell Signaling Technology). After incubated with HRP-conjugated antibody (Aspen, China) for 1 h, the membrane was incubated with Immobilon ECL substrate kit (Millipore, USA) for 1 min and then exposed to X-ray film using BioSpectrum 600 Imaging System (UVP, CA, USA).
8
Western Blot Analysis of Proteins
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Cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China). Proteins were separated by SDS-PAGE and transferred electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA). Next, the membrane was blocked in 5% non-fat milk at room temperature for 2 h, and then incubated with the primary antibodies at 4°C overnight, followed by the secondary antibody for 1 h at 37°C. Finally, the blots were detected by the Immobilon ECL substrate kit (Millipore). GAPDH was utilized as an internal control.
9
Cell Lysate Preparation and Protein Quantification
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The cell lysates were prepared with RIPA buffer (Thermo Scientific), the concentration of which was determined using a bicinchoninic acid protein assay kit (Pierce, Thermo Scientific). The immunoreactive bands were determined with an Immobilon ECL substrate kit (Millipore, Merck KGaA, Germany), while the images were acquired using a BioSpectrum 600 Imaging System (UVP, CA, USA). Detailed information regarding the primary antibodies used in this study is listed in Supplementary Table 3 .
10
Western Blot Protein Quantification
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Equal amount of total protein (30–60 μg) was separated by SDS-PAGE (Beyotime Biotechnology, China), then transferred to the PVDF membrane (Millipore, USA). Afterward, the membranes were incubated overnight with primary antibodies. Then incubated with HRP-conjugated antibody (Aspen, China) for 1 h. Afterwards membranes were incubated with Immobilon ECL substrate kit (Millipore, USA) for 1 min and detected by BioSpectrum 600 Imaging System (UVP, USA).
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