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Ecl reagent

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ECL reagent is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It catalyzes a light-emitting chemical reaction when exposed to peroxidase-conjugated antibodies, allowing for the visualization of target proteins.

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Lab products found in correlation

Pvdf membrane, by Merck Group (35 mentions) Protease inhibitor cocktail, by Roche (18 mentions) Nitrocellulose membrane, by Cytiva (16 mentions) Protease inhibitor cocktail, by Merck Group (15 mentions) Imagequant las 4000, by GE Healthcare (12 mentions) Ripa buffer, by Thermo Fisher Scientific (12 mentions) Pvdf membrane, by Bio-Rad (10 mentions) Nitrocellulose membrane, by Bio-Rad (10 mentions) Pvdf membrane, by Cytiva (10 mentions) Bca protein assay kit, by Thermo Fisher Scientific (9 mentions) β actin, by Cell Signaling Technology (8 mentions) Polyvinylidene difluoride membrane, by Merck Group (8 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (8 mentions) Ripa buffer, by Merck Group (7 mentions) X ray film, by Fujifilm (6 mentions) Ripa lysis buffer, by Merck Group (6 mentions) β actin, by Merck Group (6 mentions) Chemidoc imager, by Bio-Rad (6 mentions) Gapdh, by Cell Signaling Technology (6 mentions) Protease inhibitor, by Roche (6 mentions)

442 protocols using ecl reagent

1

Protein Isolation and Western Blot Analysis

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Crude proteins were isolated from NSCLC cells following a previously reported protocol (Wei et al. 2020 (link)). Briefly, the cells were first treated with ice-cold RIPA lysis buffer (Sigma, USA) for 15 min and then boiled for 10 min to release proteins. The denatured protein (25 μg) was loaded into 12% SDS-PAGE. Following electrophoresis, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Next, the membrane was blocked in 5% non-fat milk, followed by incubation with primary antibodies against N-cadherin (1:1000, CST, USA), EIF4A3 (1:2000, Abcam, USA), E-cadherin (1:2000, Abcam, USA), MMP9 (1:2000, Abcam, USA), Vimentin (1:2000, Abcam, USA), and GAPDH (1:2000, Proteintech, USA) overnight at 4 °C. Next, the membrane was treated with the secondary antibody (1:10,000, Jackson, USA) for 2 h at room temperature. Finally, ECL reagent (Amersham, UK) was applied to detect the protein bands. The grey value of each band was assessed with ImageJ software (National Institutes of Health, USA) (Chen et al. 2020 (link)).
Zhang Y., Qi W, & Wu Y. (2023). EIF4A3-induced circular RNA SCAP facilitates tumorigenesis and progression of non-small-cell lung cancer via miR-7/SMAD2 signaling. Environmental Science and Pollution Research International, 30(24), 65237-65249.
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2

Western Blot Analysis of Brain Proteins

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Sagittally cut hemi-brains brains were homogenized in ice-cold RIPA buffer (PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added protease inhibitors (Protease Inhibitor Cocktail P8340 and, Phosphatase Inhibitor Cocktail 1&2, Sigma-Aldrich, St. Louis, MO; Phenylmethylsulfonylfluoride (PMSF), Fluka-Biochemica, Switzerland), incubated on ice for 30 min and centrifuged at 14,000 g for 10 min at 4°C. After centrifugation, the supernatant was collected and total protein concentrations were measured using the Bradford method. Tissue lysate was added to electrophoresis sample buffer and separated by SDS/PAGE under reducing conditions on a 12% separation gel. Proteins were transferred to nitrocellulose membranes using the iBlot dry-blotting system (Invitrogen Corp., Carlsbad, CA). Unspecific binding was blocked by incubation in 5% milk blocking buffer (PBS, 5% nonfat milk and 0.1% Tween 20). Membrane bound proteins were immunoblotted with antibodies to BDNF, NGF, NT3, Nrf2 (Santa Cruz Biotech, Santa Cruz, CA), p-AMPK, AMPKα (Cell Signaling, Beverly, MA), p-Nrf2 (BIOSS Antibodies, Woburn, MA) and β-actin (Abcam Inc., Cambridge, MA). Signals were developed using ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, England). Densities of the bands were evaluated using Image J software.
Allard J.S., Perez E.J., Fukui K., Carpenter P., Ingram D.K, & de Cabo R. (2015). Prolonged metformin treatment leads to reduced transcription of Nrf2 and neurotrophic factors without cognitive impairment in older C57BL/6J mice. Behavioural brain research, 301, 1-9.
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3

Luciferase Biosensor Protein Expression

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After corresponding treatment, the transfected cells were harvested in the lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 mM deoxycholic acid and 1 mM EDTA). The cell lysates was directly subjected to SDS-PAGE and probed with a 1:1000 dilution of goat anti-luciferase polyclonal antibody (anti-luciferase pAb, Promega, catalog #G745A) to confirm the expression of the biosensor. The primary antibody was detected with a 1:2000 dilution of HRP-conjugated donkey anti-goat IgG (Promega, catalog #V805A). Blots were developed using enhanced chemiluminescence (ECL) reagent (Amersham BioSciences). Images were gathered by Alpha Innotech’s FluorChem imaging system (Alpha Innotech Corp., San Leandro, CA, USA).
Chen L., Leng W.B., Li D.Z., Xia H.W., Ren M., Tang Q.L., Gong Q.Y., Gao F.B, & Bi F. (2017). Noninvasive Imaging of Ras Activity by Monomolecular Biosensor Based on Split-Luciferase Complementary Assay. Scientific Reports, 7, 9945.
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4

Quantitative Immunoblot Analysis of Cellular Proteins

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Protein extracts were generated as described previously [23 (link)]. Equal amounts of protein (25 μg) were resolved by SDS-PAGE and subjected to immunoblot analysis. Antigen–antibody interactions were detected with ECL reagent (Amersham, Amersham, UK). Proteins were detected using the following antibodies: ER (sc-8002; Santa Cruz Biotechnology, Dallas, Texas, USA) and α-tubulin (Sigma-Aldrich, Poole, Dorset, UK). Secondary antibodies were used at concentration of 1/2000 (Dako, Denmark).
Simigdala N., Gao Q., Pancholi S., Roberg-Larsen H., Zvelebil M., Ribas R., Folkerd E., Thompson A., Bhamra A., Dowsett M, & Martin L.A. (2016). Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer. Breast Cancer Research : BCR, 18, 58.
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5

Western Blot Analysis of Kidney Proteins

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Kidney tissue was lysed with a lysis buffer (50 mM Tris-HCl[pH 7.5], 250 mM NaCl, 5 mM EDTA, 50 mM NaF, and 0.5%NP-40) containing a protease inhibitor cocktail (Boehringer Mannheim, Germany). The lysate was centrifuged at 14,000g for 20 min at 4°C. Total protein content of the supernatant was estimated by BCA assay (Pierce, Rockford, IL, USA). Proteins were separated by 4-20% SDS-PAGE, transferred to nitrocellulose membranes (Amersham Life Sciences, Arlington Heights, IL), and blocked with 5% nonfat milk in Tween 20-Tris-buffered saline for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies at 1:1000 dilutions in blocking buffer (TTBS with 2% nonfat milk). The following primary antibodies were used in the present study: β-actin (Cell Signaling, Danvers, MA), TNF-α (Abcam, Cambridge, MA), iNOS, and IL-10 (Santa Cruz, Dallas, TX). After washing, the membranes were incubated with 1:10,000 diluted horseradish peroxidase conjugated secondary antibody (Jackson Immunoresearch Lab, West Grove, PA) for 1h at room temperature, again washed, reacted with ECL reagent (Amersham Life Science, Pittsburgh, PA), and subjected to autoradiography. They were scanned and analyzed with NIH Image J software.
Samuvel D.J., Shunmugavel A., Singh A.K., Singh I, & Khan M. (2016). S-Nitrosoglutathione ameliorates acute renal dysfunction in a rat model of lipopolysaccharide-induced sepsis. The Journal of Pharmacy and Pharmacology, 68(10), 1310-1319.
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6

Quantitative Analysis of Apoptosis Regulators

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Total proteins were extracted from HepG2 cells using lysis buffer. In brief, cells were mixed with lysis buffer on ice for 30-min incubation. Ultrasonic rupture was then performed briefly, followed by centrifugation at 10 000 g for 15 min. The supernatant was transferred to a new tube and kept at −20°C for further use. In Western blotting, proteins were separated by 10% SDS-PAGE and were transferred to PVDF membrane (Pall Life, USA). Non-specific binding sites were blocked by 5% defatted milk powder for 2 h. Primary antibody against Bcl-2 (1:1 000, Cell Signaling, USA) or Bax (1:2 000, Cell Signaling, USA) was then applied for 4°C overnight incubation. On the next day, the membrane was rinsed in PBST, and was incubated with goat anti-rabbit secondary antibody (1:2 000, Cell Signaling, USA) for 30-min incubation. ECL reagent (Amersham Bioscience, USA) was used to develop the membrane, which was then exposed to X-rays. The optical density of each protein band was processed by Quantity One software. All experiments were performed in replicates (N=4).
Li S, & Zheng L. (2016). Effect of Combined Treatment Using Wilfortrine and Paclitaxel in Liver Cancer and Related Mechanism. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 1109-1114.
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7

StAR and CRTC2 Protein Analysis

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Cells were harvested and lysed with ProteoJET™ membrane protein extraction buffer (Fermentas, USA) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF), Phosphatase inhibitor (20×: 125 mM NaF, 250 mM B-glycerophosphate, 250 mM para-nitrophenyl phosphate, 25 mM NaVO3) and protease inhibitor (Sigma, P8340) were added. Extracts were quantified by BCA protein assay kit (Pierce Bio-technology, Inc., Rockford, IL, USA). Proteins were incubated with an antibody raised against recombinant mouse StAR protein (Dr. Dale Buck Hales, University of Illinois at Chicago, USA), CRTC2 (Dr. H. Takemori, National Institute of Biomedical Innovation, Osaka, Japan) for 1 h. Protein bands were visualized by ECL reagent (Amersham Biosciences) and exposing the membrane against Hyperfilm (Amersham Biosciences).
Lee J., Tong T., Takemori H, & Jefcoate C. (2015). Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB. Molecular and cellular endocrinology, 408, 80-89.
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8

Western Blot Analysis of AQP4 and GFP

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BN-PAGE gels were destained with 40% (v/v) methanol, 10% (v/v) glacial acetic acid for 30 min, refreshing the destaining solution every 10 min. Gels were soaked for 30 min in 1% SDS in Tris-buffered saline, pH 7.4, at room temperature. Proteins were blotted onto PVDF membrane by wet transfer at 100 V for 1 h. Coomassie-stained BSA marker bands were marked onto the membrane using a felt-tipped pen. Membranes were blocked in 20% (w/v) Marvel-skimmed milk powder in 0.1% PBS-Tween for 1 h. Membranes were incubated overnight at 4 °C on a roller in rabbit anti-AQP4 antibody (Abcam, ab128906) diluted 1:5,000 or rabbit anti-GFP (Abcam, ab6556) diluted 1:10,000, both in 5 ml of 0.1% PBS-Tween. Membranes were washed in 0.1% PBS-Tween and incubated with donkey anti-rabbit HRP (Santa Cruz, sc-2313) diluted 1:10,000 in 20 ml of 0.1% PBS-Tween at room temperature for 1 h. HRP was detected on x-ray film using ECL reagent (Amersham Biosciences).
Kitchen P., Conner M.T., Bill R.M, & Conner A.C. (2016). Structural Determinants of Oligomerization of the Aquaporin-4 Channel. The Journal of Biological Chemistry, 291(13), 6858-6871.
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9

Protein Expression Analysis via Western Blot

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Cells were lysed in lysis buffer (Beyotime) containing complete protease mixture (Sigma-Aldrich). After centrifugation, the lysates were boiled in SDS loading buffer and resolved by 12% acrylamide gel electrophoresis in the presence of SDS (SDS-PAGE), then transferred to a PVDF membrane (Millipore) and probed with 1:1000 dilution of a rabbit anti-p38 or anti-beta-actin polyclonal antibody (Santa Cruz Biotechnology), followed by 1:1000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Promega). Then the polypeptides were revealed using ECL reagent (Amersham Biosciences). All of the figures illustrating Western blotting analyses are representative of at least three independent experiments.
Liang X., Liu Y., Mei S., Zhang M., Xin J., Zhang Y, & Yang R. (2015). MicroRNA-22 Impairs Anti-Tumor Ability of Dendritic Cells by Targeting p38. PLoS ONE, 10(3), e0121510.
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10

Immunoblotting of Murine Immune Proteins

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Isolated white blood cell proteins from mice and rats were separated by 7.5% SDS-PAGE under reducing conditions. After blotting on a nitrocellulose membrane, immunoblotting was accomplished using antibodies against β-actin (rabbit, monoclonal, 1:2500, Sigma-Aldrich, Seelze, Germany), and iNOS (purified anti iNOS, mouse, monoclonal, 1:2500, BD Biosciences, Franklin Lakes, NJ). Detection was accomplished with either Super Signal Substrate (Pierce, Rockford, IL, USA) or ECL Reagent (Amersham, Piscataway, NJ, USA). Bands were evaluated by densitometry.
Steven S., Dib M., Roohani S., Kashani F., Münzel T, & Daiber A. (2017). Time Response of Oxidative/Nitrosative Stress and Inflammation in LPS-Induced Endotoxaemia—A Comparative Study of Mice and Rats. International Journal of Molecular Sciences, 18(10), 2176.
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