Briefly, frozen gastrocnemius muscle was homogenized in lysis buffer and protein concentration was determined by the Bradford method. Crude gastrocnemius muscle homogenates were fractionated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were stained with Ponceau red to verify equal loading and transfer. Membranes were then blocked at room temperature for 1hr in 5% non-fat milk Tris-buffered saline with 0.1% Tween-20. Primary antibodies for phosphorylated 4E-BP1 (T37/44) (cat#2855, 1:1000), total 4E-BP1 (cat#9452, 1:2000), phosphorylated rpS6 (S240/244) (cat#2215, 1:2000), total rpS6 (cat#2708, 1:2000) were incubated overnight in 5% TBST milk. Membranes were then incubated in 5% milk-TBST containing anti-rabbit (cat#7074, 1:5000) IgG horseradish-peroxidase conjugated secondary antibodies for 1h at room temperature. All antibodies were from Cell Signaling Technology. Enhanced chemiluminescence (ECL) (GE Healthcare Life Sciences, Piscataway, NJ) was used to visualize the antibody-antigen interactions. Immunoblot images were collected using a digital imager (SynGene GBox, Frederick, MD) and quantified by densitometry using imaging software (Image J; NIH).
Enhanced chemiluminescence ecl
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Enhanced chemiluminescence (ECL) is a laboratory technique used to detect and quantify specific proteins in a sample. It utilizes a chemical reaction that produces light, which is then detected and measured by specialized equipment. ECL is commonly used in western blot analysis and other protein detection applications.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lactacystin, by Merck Group (1 mentions)
Sodium pyrophosphate, by Merck Group (1 mentions)
Monosodium phosphate, by Merck Group (1 mentions)
Gliotoxin, by Merck Group (1 mentions)
Sodium fluoride, by ITW Reagents (1 mentions)
Epoxomicin, by Merck Group (1 mentions)
Hypoxanthine, by Merck Group (1 mentions)
Phenylmethylsulphonyl fluoride, by Merck Group (1 mentions)
Sorbitol, by Merck Group (1 mentions)
Mg115, by Merck Group (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
15 protocols using enhanced chemiluminescence ecl
1
Muscle Protein Signaling Analysis
2
Comprehensive Ubiquitin Assay Protocol
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Hypoxanthine, sorbitol, percoll, MG132, Epoxomicin, Lactacystin, Clasto-Lactacystin β-lactone, Gliotoxin, MG115, EDTA, sodium phosphate dibasic, monosodium phosphate, Tris HCl, sodium chloride, Tween-20, sodium pyrophosphate, glycerol-2-phosphate, saponin, bovine serum albumin (BSA), dithiothreitol (DTT) and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma. Glutathione acceptor beads, protein A donor beads, DELFIA enhancement solution and DELFIA secondary antibody (Eu-N1 rabbit Anti-mouse-IgG) came from Perkin Elmer. NP-40 was purchased from Calbiochem, sodium fluoride from Panreac, antibody anti-ubiquitin P4D1 from Santacruz, deubiquitylases inhibitor PR-619 came from Merck, complete mini EDTA protease inhibitor cocktail from Roche, antibody anti-ubiquitin FK2 from Enzo, biotin-TUBEs from Life sensor, RPMI 1640 medium from Gibco, AlbuMAX II from Invitrogen, bortezomib from Selleckchem, enhanced chemiluminescence (ECL) from GE Healthcare and PBS from Oxoid. Atovaquone was prepared in house.
3
Optimizing Protein Expression with DMEM and Lipofectamine
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Dulbecco’s modified eagle medium (DMEM), Opti-MEM, Lipofectamine 2000, penicillin-streptomycin solution, trypsin-EDTA and PCR primers were purchased from ThermoFisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was from PAA laboratories GmbH (Pasching, Austria). XCT-790 was from Sigma Chemical Co. (St. Louis, MO, USA). Triacsin C was from Enzo Life Sciences (Farmingdale, NY, USA). Polyclonal rabbit antibody anti-ACSL4 was generated in our laboratory36 (link). Monoclonal mouse anti-GAPDH and polyclonal rabbit anti-ERα antibodies were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Monoclonal mouse anti-β-tubulin was from Upstate Group Inc (Temecula, CA, USA). Polyclonal rabbit anti-ERRα chromatin immunoprecipitation (ChIP) grade was from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies and Immuno-Blot PDVF membrane were from Bio-Rad Laboratories (Hercules, CA, USA). Enhanced chemiluminescence (ECL) was from GE Healthcare (Buckinghamshire, UK). Sterile and plastic material for tissue culture was from Orange Scientific (Braine-l′Alleud, Belgium). All other reagents were of the highest grade available.
4
Protein Expression Quantification Protocol
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Anti-phospho-S6 (S235/236, 1:500), anti-S6 (1:1000), anti-phospho-4E-BP (T37/46, 1:500), anti-4E-BP (1:1000), anti-phospho-STAT3 (Y705, 1:500) and anti-STAT3 (1:500) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Actin (1:10,000) antibodies, phosphatase Inhibitor Cocktails 2 and 3, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (St. Louis, MO). The nitrocellulose membrane was purchased from EMD Millipore (Billerica, MA, USA). Enhanced chemiluminescence (ECL) was purchased from GE Healthcare (Pittsburg, PA). Donkey anti-rabbit and donkey anti-mouse horseradish peroxidase (HRP) were purchased from Jackson ImmunoResearch (West Grove, PA). AMV reverse transcriptase was purchased from Promega (Madison, WI). SYBR Green PCR Master mix was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). EDTA-free complete mini Protease Inhibitor Cocktail was purchased from Roche (Indianapolis, IN). NuPAGE Bis-Tris precast gels and Phosphate buffered saline (PBS) were purchased from Life Technologies (Grand Island, NY). Bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Rockford, IL). ProSignal Blotting Film was purchased from Genesee Scientific (El Cajon, CA). Ethyl alcohol (190 proof) was purchased from VWR (Radnor, PA).
5
Epithelial-Mesenchymal Transition Pathway Analysis
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Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin mixed solution, 0.25% trypsin, Matrigel, and Transwell chambers were obtained from Corning Life Sciences (Corning, NY, USA). Opti-MEM medium and Lipofectamine 2000 were from Invitrogen (Invitrogen, Carlsbad, CA). Hoechst 33258 was from Biyuntian Biotechnology (Shanghai, China). DMSO was from Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemiluminescence (ECL) was obtained from GE Healthcare (Pittsburgh, PA, USA). β−actin (sc-47778) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p38 (cat. no. 8690T), p-p38 (cat. no. 4511T), ERK (cat. no. 4695T), p-ERK (cat. no. 4370T), N-cadherin (13116T), Slug (9585T), P21 (2947T), CyclinD1 (2978T), and Smad3 (9523T) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
6
Protein Expression Analysis of Stem Cells
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The protein of the cells treated was extracted, and BCA Kit (Sigma-Aldrich, Germany) was used to measure the protein concentrations. Proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Danvers, MA, USA), and 5% nonfat milk was used to block PVDF membranes for 1 hour at 37°C. The dilution degrees of anti-OCT4 antibody (Abcam, USA), anti-Nanog antibody (Abcam, USA) and GAPDH (Abcam, USA) were 1:1000, 1:1000 and 1:2000, respectively. Three antibodies were hybridized overnight at 4°C and were combined with the HRP‑linked secondary antibody (BOSTER, Wuhan, China) for 2 hours at RT after PVDF membranes were washed. Finally, enhanced chemiluminescence (ECL) (General Electric Healthcare, Aurora, OH, USA) was used to visualize the bands of the proteins.
7
Immunoblotting Protein Extraction Protocol
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For immunoblotting, cells were lysed in ice-cold 10 mM Tris–HCl, pH7.5, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 100 μM Na3VO4, 2 μM ZnCl2, and 1 mM phenylmethylsulfonyl fluoride (modified Frackelton buffer). Insoluble material was removed by centrifugation (14,000×g, 10 min, 4 °C), and protein concentration was determined using a commercial Bradford assay kit according to the manufacturer’s instructions (Bio-Rad, Munich, Germany). Equal amounts of protein (20 μg per lane) were resolved by reducing SDS/PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Freiburg, Germany). Western blots were performed using specified primary antibodies and corresponding secondary horseradish peroxidase-linked polyclonal anti-rabbit antibody from abcam (Berlin, Germany). Immune complexes were visualized by enhanced chemiluminescence (ECL) from GE Healthcare Life Sciences.
8
Purification and Antibody Production for Recombinant Ia and Ib
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Recombinant Ia and Ib were purified as described earlier [10 (link)]. The Anti-Ib antibody was obtained from rabbits immunized with purified Ib [13 (link)]. Amitriptyline hydrochloride and imipramine hydrochloride were purchased from Fujifilm Wako Pure Chem (Osaka, Japan). GW4869 hydrate, bromoenol lactone (BEL), monoclonal anti-ceramide IgM monoclonal antibody (clone: 15B4) produced in mouse, beta-actin and p-nitrophenyl N-acetyl-β-d -glucosaminide were purchased from Merck (Tokyo, Japan). Rabbit anti-3-β-actin antibody and anti-acid sphingomyelinase (H-181) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Amplex Red Sphingomyelinase Assay Kit, Hanks’ balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), Alexa Fluor 488 phalloidin conjugate, 4′,6′-diamino-2-phenylindole (DAPI), Alexa Fluor 568-conjugated goat anti-rabbit IgG, Alexa Fluor 488-conjugated goat anti-rabbit IgG, and Alexa Flour 564-conjugated goat anti-mouse IgM were purchased from Thermo Fisher Sci. (Tokyo, Japan). Enhanced chemiluminescence (ECL, Saint Paul, MI, USA) kits, peroxidase-conjugated streptavidin, and horseradish peroxidase-labeled anti-rabbit IgG were obtained from GE Healthcare (Tokyo, Japan).
9
Signaling Pathways in T Cell Activation
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LY294002 (LY), oligomycin, fluoro-carbonyl cyanide phenylhydrazone (FCCP), rotenone, antimycin A, RPMI–1640 cell culture medium, fetal bovine serum (FBS) and antibody against actin were from Sigma Aldrich. Thapsigargin (TG), rapamycin, okadaic acid (OA), CAL–101 and calyculin A were from Calbiochem. OVA323-339 peptide (ISQAVHAAHAEINEAGR) was custom synthesized by Genscript (New Jersey, USA). ATP detection kit, Dynabeads CD4 T cell kit for negative selection and dynabeads pan mouse IgG were from Life Technologies. Anti-mouse CD3 (2C11), anti-mouse CD28 (37.51), anti-rat TCR (R73) and anti-Rat CD28 (JJ319) were obtained from BD Pharmingen Biosciences. Goat anti-hamster and goat anti-mouse antibodies for cross-linking were purchased from Jackson ImmunoResearch Laboratories. PP2A immuno-precipitation phosphatase assay kit was from Upstate/Millipore. Enhanced chemiluminescence (ECL) was obtained from GE Healthcare. Antibodies to phosphorylated and total AMPKα (Thr172), AMPKβ1(Ser108), Acetyl CoA carboxylase(ACC) (Ser79), S6(S235/236), 4EBP1(Thr37/46), AKT(S473) and FOXO1(T24) were obtained from Cell Signaling Technology.
10
Hepatocyte Metabolism and Mitochondrial Function
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HepG2 cells were acquired from European Collection of Cell Cultures (ECACC, Salisbury, UK). HepaRG cells, basal media, growth and differentiation additives were purchased from Biopredic International (Saint Grégoire, France). Dulbecco's modified media (DMEM) high glucose, fetal bovine serum (FBS), Cell Tracker 5- chloromethylfluorescein diacetate (CMFDA), NUPAGE 4–12% gels, rat tail collagen I and phosphate buffered saline (PBS) were purchased from Life Technologies (Paisley, UK). Nitrocellulose membrane and enhanced chemiluminescence (ECL) were purchased from GE Healthcare (Buckinghamshire, UK). All Seahorse consumables were purchased from Agilent (Santa Clara, USA). High precision pump – pump 11 was purchased from Harvard apparatus (Massachusetts, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazol carbocyanine iodide (JC-1) was purchased from Abcam (Cambridge, UK). Cytotoxicity Detection Kit was purchased from Roche Diagnostics Ltd. (West Sussex, UK). Balch homogeniser was purchased from Isobiotech (Heidelberg, Germany). Williams' E Medium powder (with l -Glutamine, without glucose) was manufactured by United States Biological. All bile acids, bile salts, MK571, bosentan were purchased from Sigma Aldrich (Dorset UK).
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