U251MG cells (105 cells/1000 µL/well) were cultured in DMEM/F12 culturing condition (serum-free medium as recently described [14 (link)]) using a 24-well plate (Corning) for 72 h. U251MG Cells were lysed in ice-cold lysis buffer containing phosphatase inhibitor (CST). Whole proteins from cultured cells were extracted using cell lysis buffer following the manufacturer’s instructions (CST). Proteins were separated by SDS-PAGE in a 10% resolving gel and electro-transferred onto polyvinylidene difluoride membranes (Merck). Membranes were blocked in 3% skim milk (GE Healthcare, Minato-ku, Tokyo, Japan) in TBS-T at ambient temperature for 1 h with agitation, and incubated with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti-RPS6 antibody (1:1000), mouse polyclonal anti-β-actin antibody (1:1000), rabbit polyclonal anti-STAT3 (1:1000), rabbit polyclonal anti-p-STAT3 (Tyr705) (1:1000), rabbit polyclonal anti-SOX2 (1:1000), and mouse monoclonal anti-Nestin (1:1000) antibodies. After three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (rabbit: GE Healthcare, mouse: GE Healthcare) with 3% skim milk for 1 h. Finally, immunoreactive protein bands were visualized by using ECL select detection reagents (GE Healthcare) and captured using the LAS4000EPUVmini (GE Healthcare).
Ecl select detection reagent
Manufactured by GE Healthcare
Sourced in United States
The ECL Select detection reagent is a chemiluminescent substrate used in Western blot analysis. It provides a sensitive and stable signal for the detection of target proteins labeled with horseradish peroxidase (HRP) conjugates.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Skim milk, by GE Healthcare (2 mentions)
Las4000epuvmini, by GE Healthcare (2 mentions)
24 well plate, by Corning (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti lamin a c, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Criterion tgx, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions)
Crizotinib, by Selleck Chemicals (1 mentions)
Anti alk antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail set 3, by Fujifilm (1 mentions)
Iblot2 system, by Thermo Fisher Scientific (1 mentions)
Ecl prime detection reagent, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
8 protocols using ecl select detection reagent
1
Western Blot Analysis of U251MG Cells
2
Western Blotting of Protein Markers
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Cells were lysed in ice‐cold lysis buffer containing phosphatase inhibitor (CST). Whole patient tissues and proteins from cultured cells were extracted using cell lysis buffer in accordance with the manufacturer's instructions (CST). Proteins were separated through SDS‐PAGE in a 10% resolving gel and electrotransferred onto polyvinylidene difluoride membranes (Merck).
Membranes were blocked in 3% skim milk (GE Healthcare) with TBS‐T at ambient temperature for 1 h with agitation and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti‐RPS6 antibody (1:1000), mouse polyclonal anti‐β‐actin antibody (1:1000), rabbit polyclonal anti‐STAT3 (1:1000), rabbit polyclonal anti‐p‐STAT3 (Tyr705) (1:1000), rabbit polyclonal anti‐SOX2 (1:1000), and mouse monoclonal anti‐Nestin (1:1000) antibodies. After three washes, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody (rabbit: GE Healthcare, mouse: GE Healthcare) with 3% skim milk for 1 h. Finally, immunoreactive protein bands were visualized using ECL select detection reagents (GE Healthcare) and detected using LAS4000EPUVmini (GE Healthcare).
Membranes were blocked in 3% skim milk (GE Healthcare) with TBS‐T at ambient temperature for 1 h with agitation and incubated with the following primary antibodies overnight at 4°C: rabbit polyclonal anti‐RPS6 antibody (1:1000), mouse polyclonal anti‐β‐actin antibody (1:1000), rabbit polyclonal anti‐STAT3 (1:1000), rabbit polyclonal anti‐p‐STAT3 (Tyr705) (1:1000), rabbit polyclonal anti‐SOX2 (1:1000), and mouse monoclonal anti‐Nestin (1:1000) antibodies. After three washes, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody (rabbit: GE Healthcare, mouse: GE Healthcare) with 3% skim milk for 1 h. Finally, immunoreactive protein bands were visualized using ECL select detection reagents (GE Healthcare) and detected using LAS4000EPUVmini (GE Healthcare).
3
Protein Extraction and Immunodetection
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Protein extraction and immunodetections were performed as previously described [62 (link)], using ECL Select detection reagent (GE Healthcare) and anti-p53 (DO-1) anti-RelA/p65 (C-20) anti- p21 (C19), anti-GAPDH (6C5) (Santa Cruz Biotechnology, Heidelberg, Germany). When appropriate, nuclear and cytoplasmic fractionation was performed. MCF7, A549 and H1299 cell lines were seeded on 100mm Petri dishes and treated at 80% confluence with Doxo, TNF⍺, BAY or the combination of the drugs for 16 hours. Cells were harvested and cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, ThermoFisher Scientific), following the instructions provided by the manufacturer. 20 μg of nuclear and cytoplasmic extracts were loaded on a 12% poly-acrylamide gel and transferred to nitrocellulose membranes. Antibodies used for detection were: anti-Histone H3 (clone #: ab1791, AbCam, Milan, Italy) and anti-Lamin A/C (clone #: 2032, Cell Signaling, Milan, Italy) used as nuclear loading control, and anti-GAPDH used as cytoplasmic loading control.
4
Quantitative Immunoblot Analysis of MAM Proteins
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The purified MAM was lysed in RIPA buffer plus the protease inhibitor. 30 μg of total proteins from the MAM were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes following protein determination. The membranes were then incubated with 5% BSA for 1.5 hours, followed by the primary antibodies against PACS2, PSS1, MFN2, Sig-1r, VAPB, and CHOP. The detailed information of the antibodies was shown in Table S1 . The secondary antibodies were then incubated for 1.5 hours. The quantitative expression level of proteins was determined using the ChemiDoc XRS+ imaging system (Bio-Rad, USA) after being detected with an ECL select detection reagent (GE Healthcare, USA).
5
Quantifying Nascent Protein Synthesis
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Using Puromycin, which incorporates into nascent peptide chains, the SUnSET assay [35 (link)] was used herein to quantify global neoprotein synthesis in cultured cells. Following 24 or 48hrs of treatment, puromycin was added to the culture media (final concentration of 1μM) for exactly 30 minutes prior to harvesting the cells in 150μl 1x Laemmli buffer (containing protease and phosphatase inhibitors; Roche) per well of a 24-well plate. Protein concentration was normalised to a constant weight/volume and 10μg protein was electrophoresed on 4–15% acrylamide gels (BioRad Criterion TGX). Proteins were wet-transferred to a nitrocellulose membrane, probed for anti-puromycin (1:1000; MerkMillipore MABE343, clone 12D10) overnight, followed by anti-mouse (1:25000; GE Health Care) for 90 minutes. Bands were visualised using ECL select detection reagent (GE Health Care) and exposing the membrane to photographic film in a dark room. Densitometry of each lane (i.e. all bands) indicated the amount of puromycin incorporated into newly synthesized proteins within a 30-minute window.
6
MTT Assay and Western Blot for ALK Inhibition
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Cell viability was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (Invitrogen). Absorbance was quantified with a ThermoFisher Spectrophotometer 1000 (Molecular Devices, Inc.) at a wavelength of 540 nm. Crizotinib (PF-02341066, Selleck Chemicals) (0.25 µM and 2 µM for H3122 and H2228 cells respectively) was used as a positive control.
Cells were lysed in buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 25 mM Tris-HCL (pH 7.4), and protease inhibitor cocktail set III (Wako Pure Chemical Industries, Japan). Proteins were separated by 9% and 11% SDS-PAGE for blotting with ALK and β-actin antibodies, respectively, and then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Proteins were detected by immunoblotting using ECL Select detection reagent (GE Healthcare, Italy). Anti-ALK antibody (Cell Signaling Technology) was used at a dilution of 1:1000. Anti-β–actin antibody (Proteintech) was used at a dilution of 1:2000.
Cells were lysed in buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid sodium salt, 0.1% SDS, 25 mM Tris-HCL (pH 7.4), and protease inhibitor cocktail set III (Wako Pure Chemical Industries, Japan). Proteins were separated by 9% and 11% SDS-PAGE for blotting with ALK and β-actin antibodies, respectively, and then transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). Proteins were detected by immunoblotting using ECL Select detection reagent (GE Healthcare, Italy). Anti-ALK antibody (Cell Signaling Technology) was used at a dilution of 1:1000. Anti-β–actin antibody (Proteintech) was used at a dilution of 1:2000.
7
Ishikawa Cell Protein Extraction and Western Blot
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Soluble proteins from Ishikawa cell lysates were extracted as described previously (36 (link)), followed by western blot analysis with the aforementioned primary antibodies (1:1,000) at 4°C overnight. Bands were detected using the BioRad Blotting system (BioRad Laboratories, Inc., Hercules, CA, USA) with the ECL Select Detection Reagent (GE Healthcare, Little Chalfont, UK).
8
Western Blot Analysis of Phosphorylated Smad2/3
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Cells were extracted in lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Nacalai Tesque) for 1 hour on ice. The lysates were centrifuged at 10,000 rpm for 10 minutes, incubated at 70°C for 10 minutes, and separated by SDS-PAGE (30 μg protein/lane). After transferring to a PVDF membrane using the iBlot 2 system (Life Technology, Carlsbad, CA, USA), the blot was probed with the antibodies described above at 4°C overnight. After 1-hour incubation with an HRP-labeled anti-rabbit immunoglobulin antibody (Cell Signaling Technology) (diluted at 1 : 20,000) at room temperature, signals were visualized using the ECL Select detection reagent (for phosphorylated Smad2/3) (GE Healthcare, Chicago, IL, USA) or ECL Prime detection reagent (for Smad2/3 and β-actin) (GE Healthcare) and analyzed by ImageQuant LAS 500 (GE Healthcare).
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