The cells were lysed in buffer containing 50 mM Tris-HCl (pH 6.9; Sigma), 2% sodium dodecylsulfate (SDS; Nacalai), 6% 2-mercaptoethanol (Sigma), and 10% glycerol. Protein samples (20 μg) were subjected to 4%–20% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto an Immuno-Blot PVDF membrane (Bio-Rad). The membrane was treated with a mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology) at a dilution of 1 : 1000 or goat polyclonal anti-MEST antibody (Abcam) at a dilution of 1 : 200, followed by biotinylated anti-mouse IgG (Nichirei Biosciences, Inc., Tokyo, Japan) or anti-goat IgG (Nichirei) as appropriate. After the membrane was washed thoroughly, the reactive bands were visualized using the ECL Select western blotting detection system (GE healthcare, Buckinghamshire, UK) and Image Quant LAS 500 (GE).
Ecl select western blotting detection system
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The ECL Select Western Blotting Detection System is a chemiluminescent detection kit designed for the visualization of protein bands in western blotting experiments. The system utilizes an enhanced chemiluminescent (ECL) substrate to produce a luminescent signal that can be detected using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000, by Cytiva (4 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Anti hif 1α rabbit polyclonal antibody, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
Goat polyclonal anti mest antibody, by Abcam (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Tris hcl, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Mouse anti β actin monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Biotinylated anti mouse igg, by Nichirei Biosciences (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
M per lysis buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Antibody against β actin, by BioLegend (1 mentions)
Sab5200111, by Merck Group (1 mentions)
Hybond p pvdf membrane, by GE Healthcare (1 mentions)
Anti β actin polyclonal antibody, by Abcam (1 mentions)
Ab116028, by Abcam (1 mentions)
12 protocols using ecl select western blotting detection system
1
Protein Expression Analysis by Western Blot
2
Western Blot Protein Extraction and Analysis
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M-PER lysis buffer (Thermo Fisher Scientific, Inc.) was added to the cells cultured in a 6-well plate, and cells were incubated for 5 min at room temperature, with gentle agitation. The lysate was collected and transferred to a microcentrifuge tube and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was collected and transferred to a new tube for analysis. Protein concentration was determined using the bicinchoninic acid (BCA) assay. The purified protein (10 µg per lane) were subjected to 10% SDS-PAGE, and the proteins were transferred onto polyvinylidene difluoride membranes (Immobilon P; Merck Millipore), which were then probed with primary antibodies at 4°C overnight. The membranes were subsequently washed with TBS containing Tween 20, and were incubated with secondary antibodies for 1 h at room temperature with agitation. Proteins of interest were visualized with enhanced chemiluminescence (ECL) reagents using the ECL, ECL-Prime, or ECL-Select Western Blotting Detection system (Amersham; GE Healthcare Life Sciences, Chalfont, UK). Densitometry was performed using ImageJ version 1.48 (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated 3 times.
3
Smad1 Regulation by TGF-β Receptors
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HepG2 cells were transiently transfected with 6Myc-tagged Smad1 with or without expression vectors for the type I receptors and type II receptors of the TGF-β family. At 4 h post-transfection, cells were exposed to LDN-193189 for 48 h. Immunoprecipitation and Western blotting were performed as described previously64 (link). The immunoreactive proteins were visualized using an ECL Select Western blotting detection system (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s protocol.
4
Uremic Solute-Induced Cellular Stress
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Cells were treated with uremic solutes as described above for 5 hours. After treatment, the cells were lysed with RIPA Lysis Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies against phosphorylated p53 (at Ser15) and Chk1 (at Ser345) were used (both are supposed to be active forms and were purchased from Cell Signaling Technology). Chemiluminescent signals generated using the ECL Select Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) were detected by Light-Capture II Cooled CCD Camera Systems (ATTO, Tokyo, Japan). Antibody against β-actin was used as loading control (Biolegend, San Diego, CA, USA).
5
Western Blot Analysis of AQP3 Protein
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Cells were lysed in RIPA buffer (supplemented with a protease and phosphatase inhibitor cocktail) and homogenates were solubilized in Laemmli buffer [61 (link)]. 15–30 μg solubilized proteins were subjected to 12.5% SDS-polyacrilamide gel electrophoresis and transferred to the Hybond-P PVDF Membrane (GE Healthcare, Chicago, IL, USA) by electroelution. The membranes were incubated overnight with anti-AQP3 antibody produced in rabbit (SAB5200111; Sigma, Italy) diluted 1:1000 in TBS and 0.1% Tween-20. The membranes were washed and incubated for 1 h with goat anti-rabbit IgG antibody, peroxidase conjugated (AP132P; Millipore, Burlington, MA, USA) diluted 1:100,000 in blocking solution. The bands were detected with ECL™ Select western blotting detection system (GE Healthcare). Pre-stained molecular weight markers (ab116028, Abcam) were used to estimate the molecular weight of the bands. Blots were stripped and re-probed with RabMAb anti β-2-microglobulin antibody ([EP2978Y] ab75853; Abcam, 1:10000) or with anti β-actin polyclonal antibody (Cat.n. AB-81599; Immunological sciences, USA, 1:2000) as housekeeping.
6
Protein Interaction Analysis Using Immunoprecipitation
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Cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, and 1 mM PMSF) or RIPA buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1 mM PMSF) containing Complete Protease Inhibitor Cocktail (Roche). Cell lysates were spun down at 12,000 rpm for 10 min, and 50 µg supernatants were resolved on SDS-PAGE and blotted with the indicated antibodies. The amount of β-actin was used as the loading control. Blots were developed with an ECL Select Western Blotting Detection system (GE Healthcare) and imaged with the Minichemi machine (Sagecreation). Quantification of Western blots was performed using ImageJ software.
To test the direct interaction of two proteins tagged with different epitopes, the corresponding expression vectors were cotransfected in HEK293 cells. 48 h later, cells were lysed in regular IP buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM PMSF, and 1% glycerol) containing Complete Protease Inhibitor Cocktail. Cell lysates were spun at 12,000 rpm for 10 min at 4°C, and the supernatants were incubated with Flag antibody (M2)–conjugated or HA antibody–conjugated beads (∼5 µg antibody for each sample) overnight at 4°C. The beads were centrifuged and washed three times with IP buffer. Precipitated proteins were resolved by SDS-PAGE, blotted, and probed with different antibodies.
To test the direct interaction of two proteins tagged with different epitopes, the corresponding expression vectors were cotransfected in HEK293 cells. 48 h later, cells were lysed in regular IP buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, 1 mM PMSF, and 1% glycerol) containing Complete Protease Inhibitor Cocktail. Cell lysates were spun at 12,000 rpm for 10 min at 4°C, and the supernatants were incubated with Flag antibody (M2)–conjugated or HA antibody–conjugated beads (∼5 µg antibody for each sample) overnight at 4°C. The beads were centrifuged and washed three times with IP buffer. Precipitated proteins were resolved by SDS-PAGE, blotted, and probed with different antibodies.
7
Western Blot Analysis of Osteogenic Markers
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HBMScs were lysed using mammalian protein extraction RIPA reagent (Yeasen). Equivalent amounts (50 μg) of cell protein lysates were electrophoresed on an 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with primary antibodies of RUNX (1:750, ab76956, Abcam, Cambridge, MA, USA), BGLAP (1:1000, ab13421, Abcam), SSP1 (1:750, ab69498, Abcam), and GAPDH (1:1000, 60004-1-Ig, ProteinTech Group, Chicago, IL, USA). After incubating with HRP-associated second antibodies, protein blots were observed using ECL Select Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and the results were quantified using Image Pro Plus 6.0 (Media Cybernetics).
8
Glycoprotein Detection by ConA Blotting
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Total protein samples were prepared as described above. Proteins in the supernatant were separated by SDS-PAGE in a 12% slab gel and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) using an electroblot apparatus (model 200/2.0, Bio-Rad). The membrane was incubated for 1h in PBSCT buffer (138mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, 1mM MgCl2, 1mM CaCl2, 0.5% Tween 20, pH 7.2) and then for 1h in PBSCT buffer containing 0.1 μg ml–1 ConA conjugated to horseradish peroxidase (Sigma, St Louis, MO, USA). The membrane was washed five times for 5min with PBSCT. ConA-binding glycoproteins were detected with the ECL Select Western Blotting Detection System (GE Healthcare). The ConA-specific bands were quantified using Image J software.
9
Protein Extraction and Western Blotting
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Protein extraction was performed using lysis buffer [10% glycerol, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 400 mM NaCl, 0.5% NP40, 4 μg/ml aprotonin, PMSF, proteasome inhibitor MG-132 and 1 mM DTT]. Total protein (10 μg) was electrophoresed on a 10% polyacrylamide gel, and then electroblotted at 300 mA for 90 min on a nitrocellulose membrane (Invitrogen, Carlsbad, CA, USA). Western blot analysis was used to confirm the protein expression of HIF-1α and HSC70: These proteins were detected using anti-HIF-1α rabbit polyclonal antibody (1:1,000) (Cell Signaling Technology, Cat. no. 3716) and anti-HSC70 mouse monoclonal antibody (1:1,000) (Santa Cruz Biotechnology, Cat. no. sc-7298). HSC70 expression was used as a loading control. The signals were detected using the ECL Select Western Blotting Detection System (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and Image Quant LAS 4000 software (GE Healthcare Life Sciences).
10
Western Blot Analysis of Dental Stem Cells
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Proteins were collected from DPSCs using Pierce RIPA buffer (Invitrogen). The proteins were subjected to 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subsequently transferred onto Immune-Blot PVDF membranes (Bio‐Rad Laboratories). After blocking with TBST containing 2% BSA or 5% dry skim milk (Yukijirushi, Tokyo, Japan) for 1 h at room temperature, the membranes were reacted with the anti-TH antibody (1:1000) or anti-β-actin antibody (1:1000) overnight at 4 °C. Then, the membranes were stained with biotinylated anti‐rabbit IgG (Nichirei Biosciences) or anti-mouse IgG (Nichirei Biosciences) and reacted with an avidin-peroxidase conjugate (1:2000, Sigma-Aldrich). Reactive bands were detected using the ECL select Western blotting detection system (GE Healthcare, Buckinghamshire, UK). Images were analyzed by Image Quant LAS 4000 (GE Healthcare) and ImageJ 1.53e (Java 1.8.0_172, http://imagej.nih.gov/ij ).
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