To study the effect of drugs on protein expression, tissues and cells were lysed in RIPA buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 10% glycerol) containing protease and phosphatase inhibitors (Roche Diagnostics, Laval, QC, Canada). The cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. Membranes were then developed using an enhanced chemiluminescence (ECL) reagent (GE Healthcare, Piscataway, NJ, USA). α-tubulin was used as a loading control. Antibodies used for western blotting included anti‒epidermal growth factor receptor (EGFR), anti‒fibroblast growth factor receptor (FGFR)-3, and anti‒insulin-like growth factor-1 receptor (IGF-1R; Cell Signaling Technology, Beverly, MA, USA); anti‒human epidermal growth factor receptor (HER)-2, anti‒HER-3, and anti-MET (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-α-tubulin (Sigma-Aldrich); and horseradish peroxidase-conjugated secondary anti-rabbit and anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) antibodies. Membranes were then developed using the ECL reagent (GE Healthcare), and α-tubulin was used as a loading control. Quantification was performed with a Molecular Imager ChemiDoc XRS+ Imaging System (Bio-Rad, Hercules, CA, USA)
Ecl reagent
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany, China, Sweden, Brazil, Japan, Canada, France
ECL reagent is a luminescent detection solution used in Western blot analysis. It is designed to facilitate the visualization of target proteins that have been labeled with antibodies and detected using a chemiluminescent substrate. The reagent emits light upon reaction with the labeled antibodies, allowing for the sensitive detection and quantification of proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (57 mentions)
Ripa lysis buffer, by Beyotime (20 mentions)
Nitrocellulose membrane, by GE Healthcare (20 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Protease inhibitor cocktail, by Roche (17 mentions)
β actin, by Merck Group (15 mentions)
Protease inhibitor cocktail, by Merck Group (14 mentions)
Pvdf membrane, by GE Healthcare (12 mentions)
Nitrocellulose membrane, by Bio-Rad (12 mentions)
Pvdf membrane, by Thermo Fisher Scientific (12 mentions)
Protease inhibitor, by Roche (11 mentions)
β actin, by Cell Signaling Technology (10 mentions)
Bradford assay, by Bio-Rad (10 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Polyvinylidene difluoride membrane, by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Image lab software, by Bio-Rad (8 mentions)
Pvdf membrane, by Bio-Rad (8 mentions)
Quantity one software, by Bio-Rad (8 mentions)
Bca protein assay, by Thermo Fisher Scientific (8 mentions)
617 protocols using ecl reagent
1
Analyzing Protein Expression with Western Blotting
2
Western Blot Analysis of Breast Cancer Markers
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Cells were collected four to seven days post-transfection and lysed in sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
[13 ]. Cell lysates were separated
by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were
blocked with 5% fat-free milk powder in 150 mM NaCl, 50 mM Tris-HCL pH 8.0, 0.1%
Tween-20 (TBST) and incubated overnight at 4°C with the following antibodies: TERT
(Y182), PI3K p110α (04-399), ΔN-p63 (p40 [5 (link)]-[17 ] PC373),
GATA3 (09-076) (Millipore); BMI1 (D20B7), keratin 18 (DC10), cyclin D1 (DCS6), myc
(D84C12), AKT (9272), phospho-AKT-T308 (4056/244 F9), ERBB3 (4754) (Cell Signaling
Technology); FOXA1 (Ab55-178) (Abcam); AGR2 (1C3), tubulin (B-5-1-2) (Sigma);
keratin 14 (LL002, gift from Birgit Lane); ERα (Ab-16, RB-1493)
(ThermoScientific); p53 (D01, gift from David Lane). After three washes in TBST,
bound primary antibodies were detected by incubation with HRP-conjugated
anti-rabbit, anti-mouse or anti-goat IgG (GE Healthcare) at room temperature for
1 hour, washed again in TBST, and visualized using ECL reagents (GE Healthcare).
Images were captured on a Fusion FX7 scanner (Vilber Lourmat) or Hyperfilm (GE
Healthcare).
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
[13 ]. Cell lysates were separated
by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were
blocked with 5% fat-free milk powder in 150 mM NaCl, 50 mM Tris-HCL pH 8.0, 0.1%
Tween-20 (TBST) and incubated overnight at 4°C with the following antibodies: TERT
(Y182), PI3K p110α (04-399), ΔN-p63 (p40 [5 (link)]-[17 ] PC373),
GATA3 (09-076) (Millipore); BMI1 (D20B7), keratin 18 (DC10), cyclin D1 (DCS6), myc
(D84C12), AKT (9272), phospho-AKT-T308 (4056/244 F9), ERBB3 (4754) (Cell Signaling
Technology); FOXA1 (Ab55-178) (Abcam); AGR2 (1C3), tubulin (B-5-1-2) (Sigma);
keratin 14 (LL002, gift from Birgit Lane); ERα (Ab-16, RB-1493)
(ThermoScientific); p53 (D01, gift from David Lane). After three washes in TBST,
bound primary antibodies were detected by incubation with HRP-conjugated
anti-rabbit, anti-mouse or anti-goat IgG (GE Healthcare) at room temperature for
1 hour, washed again in TBST, and visualized using ECL reagents (GE Healthcare).
Images were captured on a Fusion FX7 scanner (Vilber Lourmat) or Hyperfilm (GE
Healthcare).
3
Protein Expression Analysis by Western Blotting
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Tissue protein lysates were prepared with RIPA buffer containing a cocktail of protease inhibitors and then quantified using a Protein Assay Kit (Bio-Rad). Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking in TBST solution containing 5% skim milk, membranes were incubated overnight at 4°C with specific antibodies (S9 Table ). Expression signals of each protein were detected using ECL reagents (GE Health Care) with secondary antibodies. Luminescent densities were measured using a LAS-3000 Luminescent Image Analyzer System (Fujifilm).
4
Western Blot Analysis of Cytoskeletal Proteins
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Protein samples were extracted using Nonidet P-40 buffer. SDS-PAGE was performed on 5–13% acrylamide gels. Proteins were electrotransferred to nitrocellulose membrane and probed with primary antibodies. The antibodies used included mouse anti-α-SMA (Acris Antibodies, Germany), molecular weight 42 kDa, and mouse anti-β-actin (Sigma, USA), molecular weight 42 kDa, which served as a housekeeping reference. The membranes were incubated with the corresponding peroxidase-conjugated secondary antibodies, washed, and then incubated with ECL reagents (GE Healthcare Europe GmbH; Freigburg; GE) before exposure to high-performance chemiluminescence films. Gels were calibrated using Bio-Rad standard proteins (Hercules, CA) with markers covering a 7–240-kDa range.
Films were scanned by using image-editing software NIH ImageJ software for densitometric analysis of immunoreactive bands.
Films were scanned by using image-editing software NIH ImageJ software for densitometric analysis of immunoreactive bands.
5
FOXM1 Knockdown in OSCC Cells
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OSCC cells were plated in six-well plates and FOXM1 was knocked down. The cells were collected, suspended in lysis buffer (50 mmol/l Tris-HCl, pH 6.8, 100 mmol/l DTT, 2% SDS, 0.1% bromophenol blue and 10% glycerol) on ice for 5–10 min and centrifuged at 4°C (12,000 × g, 15 min). The protein concentrations were determined using a bicinchoninic acid protein assay. The 50 µg protein samples per lane were separated by 10% SDS-PAGE and transferred onto nitrocellulose filter membranes. The membranes were blocked in freshly prepared PBS containing 5% non-fat dried milk for 2 h at room temperature. The blots were subsequently probed with primary antibodies specific for FOXM1, E-cadherin, vimentin and β-actin (1:1,000; ab55006, ab1416, ab45939 and ab8226 respectively; Abcam,) overnight at 4°C. The membranes were washed three times with PBS-0.05% Tween 20 and incubated with the corresponding secondary antibodies (1:1,000; ab6940; Abcam) for 1 h at room temperature. The blots were subsequently incubated in the dark for enhanced chemiluminescence and visualized through exposure to ECL reagents (GE Healthcare, Chicago, IL, USA).
6
Protein Extraction and Western Blot
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Cells were harvested and lysed in 1 × RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail. The protein concentrations of each extracted protein sample were measured using Bio-Rad protein assay reagents. A total of 30 micrograms of each protein sample was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% skim milk and then incubated with specific primary antibody overnight. The antibody-probed membrane was then washed 6 times with 1 × PBST (0.05% Tween 20 in 1 × PBS). After washing, the appropriate horseradish peroxidase-labeled secondary antibodies were added to the membrane for 1 h, and then washed with 1 × PBST. The bound antibodies were detected by enhanced chemiluminescence (ECL) reagents (GE Healthcare Life Sciences; Uppsala, Sweden). The blot signals were visualized by X-ray film (Roche Applied Science, Mannheim, Germany). The intensities of signals were quantified by GeneTools software (SYNGEN, Cambridge, UK).
7
Epigenetic Regulation Analysis
8
Quantitative Immunoblot Analysis of mTOR Signaling
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Equal amounts of protein were analyzed in duplicate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following monoclonal antibodies were used: anti–phospho-Akt (Ser473, Cell signaling, Danvers, MA), anti–total Akt (Cell signaling), anti-phospho-mTOR (Ser2448, Cell signaling), anti-phospho-mTOR (Thr2446, Millipore, Bedford, MA), anti-total mTOR (Cell signaling), anti-HIF-1α (Cayman Chemical, Ann Arbor, MI), anti-VHL (Cell signaling), anti-phospho-AMPK (Thr172, Cell signaling), anti-total AMPK (Cell signaling) anti-phospho-P70S6K (Thr389, Cell signaling), anti-total P70S6K (Cell signaling), anti-phospho-4E-BP (Thr37/46, Cell signaling), anti-lactate dehydrogenase A (LDHA) (Cell signaling), anti-hexokinase-2 (HK-2, SantaCruz Biotechnology, Dallas, TX), anti-phospho-TSC2 (Ser1387) (Cell signaling), anti-total TSC2 (Cell signaling), and anti-β-actin (Bethyl, Montgomery, TX, US). Immunoreactive proteins were detected by horseradish peroxidase–conjugated secondary antibodies and enhanced using chemiluminescence (ECL) reagents (GE Healthcare Life Sciences, Piscataway, NJ). All immunoblots were performed with triplicate and visualized by LAS image analyzer (Fujifilm, Tokyo, Japan). The band density was quantified using MultiGauge (Fujifilm).
9
Macrophage Inflammatory Pathway Analysis
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Macrophages (1 × 107 cells) were seeded into a six-well plate and cultured for 24 h. After 2 h preincubation with or without anti-MaMR CTLD4-8 IgG (4.8 μg/ml), COS6 (50 μg/ml) was added, giving a final volume of 1 ml. Incubated for 30 min, the cells were collected and lysed with a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing proteinase inhibitors on ice. The protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, China). Equal amounts of total protein were separated by 8%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes (Millipore, Germany). The membranes were blocked in fresh 5% BSA dissolved in Tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 1 h, then incubated with antibody TLR4 (1:500 dilution; HuaBio, China), TLR2 (1:500; HuaBio, China), and β-tubulin (1:4,000; Abclonal, China) overnight at 4°C. They were then washed three times with TBST buffer and incubated with HRP-conjugated goat anti-rabbit IgG (Abclonal, China) for 1 h at room temperature. Immunodetection was performed using enhanced chemiluminescence (ECL) reagents (GE Healthcare, USA).
10
Analyzing Protein Signaling in Cells
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Proteins in cell lysates were detected with antibodies against phospho-PAK1 (Santa Cruz Biotechnology), PAK1, phospho-AKT, AKT, HIF1α (BD Biosciences, North Ryde, Australia), and GAPDH. Antibodies were from Cell Signalling Technology (Arundel, Australia), unless otherwise stated. Bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, UK), and the density of each band was analysed using Multigauge computer software (Berthold, Bundoora, Australia). HIF1α expression was determined in cells cultured under normoxia or hypoxia (1 % O2).
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