Ecl plus detection kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

The ECL Plus detection kit is a chemiluminescent detection system designed for western blot analysis. The kit provides reagents for the detection of proteins tagged with horseradish peroxidase-conjugated secondary antibodies.

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Lab products found in correlation

Donkey anti goat igg hrp, by Agilent Technologies (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Quantity one imaging system, by Bio-Rad (1 mentions) Bicinchoninic acid kit, by Beyotime (1 mentions) Bis tris sds page, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Dc protein assay kit, by Bio-Rad (1 mentions) Donkey anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail solution, by GenDEPOT (1 mentions) Sample buffer, by Bio-Rad (1 mentions) Radioimmunoprecipitation assay (ripa), by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp conjugated anti rabbit secondary antibody, by Abcam (1 mentions) Trypan blue, by Thermo Fisher Scientific (1 mentions) 2 7 dichlorofluorescin diacetate dcfh da, by Merck Group (1 mentions) Tris buffered saline (tbs), by Boston BioProducts (1 mentions) Dulbecco s modified eagle s medium dmem, by Atlanta Biologicals (1 mentions) Zinc oxide nanoparticles zno nps, by Merck Group (1 mentions) Titanium dioxide nanoparticles tio2 nps, by Merck Group (1 mentions)

Close spelling variants of this product

ECL kit (865 citations) ECL detection kit (265 citations) ECL Plus (212 citations) ECL Plus kit (106 citations) PLUSTM (3 citations)

22 protocols using ecl plus detection kit

Quantification of Protein Expression in Breast Cancer

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To detect protein expression levels, breast cancer cells were lysed in ice-cold RIPA lysis buffer (Biospes). Homogenates were then centrifuged at 12,000 × g for 20 min at 4°C. Protein content in the clear supernatant was quantified using a bicinchoninic acid kit (Beyotime), and samples were then reduced and stored at −80°C until use. Samples (20–50 μg protein/lane) were separated by SDS-PAGE. Proteins were transferred from the gel onto a PVDF membrane. After protein transfer, PVDF membranes were blocked with a solution containing Tris-buffered saline, 0.1% Tween 20 (TBST) and 5% fat-free milk for 2 h at room temperature. Membranes were then incubated with primary antibodies (See Table S1) overnight at 4°C. Afterwards, blots were treated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (1:10,000) in TBST containing 1% (w/v) BSA for 60 min at room temperature while shaking, and immune complexes were detected using an ECL plus detection kit (Thermo). Finally, PVDF membranes were scanned using the Quantity One Imaging system (Bio-Rad). The relative expression of target protein was normalized to that of GAPDH or β-actin. Every experiment was repeated three times.

Tan J., Zhang X., Xiao W., Liu X., Li C., Guo Y., Xiong W, & Li Y. (2019). N3ICD with the transmembrane domain can effectively inhibit EMT by correcting the position of tight/adherens junctions. Cell Adhesion & Migration, 13(1), 203-218.

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Western Blot Analysis of nNOS in Spinal Cord

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Dorsal spinal cord tissues at the L5 and L6 levels were removed, dissected, and homogenized in 300 μl RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM NaF in the presence of the protease inhibitor cocktail. Samples were then put on ice for 30 min with shaking. Lysates were centrifuged at 13,000 g for 30 min at 4°C and the supernatant was collected. The protein concentration was quantified using a DC protein assay kit (Bio-Rad, Hercules, CA). Thirty μg of total proteins of each sample was loaded and separated on 4–12% Bis-Tris SDS-PAGE (Invitrogen). The resolved proteins were transferred to nitrocellulose membranes. The membranes were treated with 5% bovine serum albumin in Tris buffer containing Tween 20 for 2 h and then incubated with a rabbit anti-nNOS antibody (Cat. #07-571-I, EMD Millipore) overnight at 4°C. The membrane was washed three times and then incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The protein band was revealed with an ECL Plus detection kit (ThermoFisher, Rockfort, IL), and the protein band intensity was quantified by using the ImageJ software program. The amounts of proteins were normalized by GAPDH (Cell Signaling Technology), which was used as a protein loading control.

Chen S.R., Jin X.G, & Pan H.L. (2017). Endogenous Nitric Oxide Inhibits Spinal NMDA Receptor Activity and Pain Hypersensitivity Induced by Nerve Injury. Neuropharmacology, 125, 156-165.

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UVB-Induced DNA Damage Assay in HaCaT Cells

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HaCaT cells were seeded (1 × 10⁶ cells/plate) in glass plates and incubated overnight. Cells were treated with different sizes (10, 20, 40, 60 and 100 nm) of silver nanoparticles at a concentration of 1 μg/mL. After 3 h, cells were irradiated with UVB (40 mJ/cm²) using a Daavlin Research Irradiator (Bryan, OH) equipped with four UVB lamps and an electronic controller to regulate UVB dosage. A majority of the wavelengths of UVB radiation were in the 280–320 nm range. Genomic DNA (treated or untreated) was isolated using DNAzol, as per manufacturer’s instructions, and transferred (500 ng) to a positively charged nitrocellulose membrane. Immobilized DNA was fixed by baking the membrane for 30 min at 80 °C. To avoid non-specific binding, a blocking buffer (5 % non-fat dry milk, 1 % Tween-20 in 20 mM TBS, pH 7.6) was utilized. Subsequently, the membrane was incubated, with an anti-CPDs antibody, for 2 h at room temperature, followed by washing with TBST (Tris buffered saline with 0.05 % Tween 20) and incubation with HRP-conjugated secondary antibody. The membrane was processed with an ECL plus detection kit (Thermo Scientific, Logan, UT) and the signal was detected using an LAS-3000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).

Palanki R., Arora S., Tyagi N., Rusu L., Singh A.P., Palanki S., Carter J.E, & Singh S. (2015). Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes. BMC Cancer, 15, 636.

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Western Blot Analysis of HENMT1 in Testes

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Proteins were extracted from 10 week-old adult testes (n = 3/genotype) using NP-40 buffer (Fluka). 20μg of protein was separated on a 12% SDS-PAGE gel, proteins transferred to PVDF membranes and then probed using HENMT1 and actin (Sigma Aldrich) antibodies. Bound antibody was detected using donkey anti-goat IgG HRP (Dako) secondary antibody and donkey anti-rabbit IgG HRP respectively and detected with enhanced chemiluminescence (ECL Plus) detection kit (Thermo Scientific).

Lim S.L., Qu Z.P., Kortschak R.D., Lawrence D.M., Geoghegan J., Hempfling A.L., Bergmann M., Goodnow C.C., Ormandy C.J., Wong L., Mann J., Scott H.S., Jamsai D., Adelson D.L, & O’Bryan M.K. (2015). HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse. PLoS Genetics, 11(10), e1005620.

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Aromatase Protein Expression Analysis

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Protein from the cells exposed to EGb761 (10 and 100 μg/mL) was extracted with radioimmuno-precipitation assay (RIPA; Thermo Scientific, Rockford, IL, USA) buffer containing 1% protease inhibitor cocktail solution (GenDEPOT, Houston, TX, USA). Lysate proteins (50 μg) denatured with 2x sample buffer (Bio-RAD, Hercules, CA, USA) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose blotting membranes. Immunodetection was performed using anti-aromatase (1:1000, abcam35604, Abcam Plc, Cambridge, UK) and anti-actin antibodies (1:4000, 13E5, Cell Signaling Technology, Danvers, MA, USA), respectively. The corresponding anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody (Abcam Plc) was used. An ECL^plus detection kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) provided the chemiluminescence substrate for HRP, and the targeted protein was visualized by autoradiography.

Kim M., Park Y.J., Ahn H., Moon B., Chung K.H, & Oh S.M. (2016). The effects of the standardized extracts of Ginkgo biloba on steroidogenesis pathways and aromatase activity in H295R human adrenocortical carcinoma cells. Environmental Health and Toxicology, 31, e2016010.

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Nanoparticle Cytotoxicity Assessment Protocol

Cited 1 time

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Fetal-bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were procured from Atlanta Biologicals (Lawrenceville, GA) and Thermo Scientific (Logan, UT), respectively. Penstrep (50X) and trypsin-EDTA were obtained from Invitrogen (Carlsbad, CA). Silver nitrate, sodium borohydride, sodium citrate tribasic dehydrates, 2’, 7’-dichlorofluorescin diacetate (DCFH-DA), Zinc-oxide nanoparticles (ZnO-NPs) and titanium-dioxide nanoparticles (TiO2-NPs) were purchased from Sigma-Aldrich (St. Louis, MO). Silver nanoparticles (Ag-NPs) were synthesized and characterized as described earlier [18 (link)]. Countess cell counting chamber slides and trypan blue were obtained from Life technologies (Grand Island, NY) and Thermo Scientific, respectively. Tris buffered saline (TBS) and Tween 20 were from Boston Bioproducts (Ashland, MA). Cell proliferation reagent (WST-1) and DNAzol was procured from Roche Diagnostics (Mannheim, Germany) and Life Technologies, respectively. ECL plus detection kit was obtained from Thermo Scientific. Following antibodies were used: Cyclobutane pyrimidine dimer (Kamiya Biomedical Company, Seattle, WA), (6-4) photoproducts (Cosmo Bio Co. Ltd, Tokyo, Japan) and 8 hydroxyguanosine (Abcam, Cambridge, MA). All respective horseradish peroxidase-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Dallas, Texas).

Tyagi N., Srivastava S.K., Arora S., Omar Y., Ijaz Z.M., AL-Ghadhban A., Deshmukh S.K., Carter J.E., Singh A.P, & Singh S. (2016). Comparative analysis of the relative potential of silver, zinc-oxide and titanium-dioxide nanoparticles against UVB-induced DNA damage for the prevention of skin carcinogenesis. Cancer letters, 383(1), 53-61.

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Detecting UV-Induced DNA Damage

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Genomic DNA from HaCaT cells (treated or untreated) was isolated using DNAzol and transferred (500 ng) to a positively-charged nitrocellulose membrane. Immobilized DNA was fixed by baking the membrane for 30 minutes at 80 °C followed by blocking (5 % non-fat dry milk in TBST) to avoid non-specific binding. Subsequently, membrane was incubated with anti-cyclobutane pyrimidine dimers (CPDs) antibody for 2 h at room temperature, washed with TBST and incubated with HRP-conjugated secondary antibody. The membrane was processed with ECL plus detection kit (Thermo Scientific) and signal detected using an LAS-3000 image analyzer (Fuji Photo Film Co., Tokyo, Japan).

Arora S., Tyagi N., Bhardwaj A., Rusu L., Palanki R., Vig K., Singh S.R., Singh A.P., Palanki S., Miller M.E., Carter J.E, & Singh S. (2015). Silver nanoparticles protect human keratinocytes against UVB radiation-induced DNA damage and apoptosis: potential for prevention of skin carcinogenesis. Nanomedicine : nanotechnology, biology, and medicine, 11(5), 1265-1275.

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Testis Protein Extraction and Western Blot

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Proteins were extracted from whole testis homogenates and isolated germ cell populations using RIPA buffer (50mM Tris-HCL; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate; 0.9% NaCl; 5mM EDTA ph 7.4) plus protease inhibitor cocktail (Calbiochem). Extracted protein was separated on a 12% SDS-PAGE gel, transferred to PVDF membranes and probed using primary antibodies. Bound antibody was detected using donkey anti-goat IgG HRP and donkey anti-rabbit IgG HRP (Dako) secondary antibodies with enhanced chemiluminescence ECL Plus detection kit (Thermo Scientific) or Clarity Max ECL substrate (BioRad).

Dunleavy J.E., Okuda H., O’Connor A.E., Merriner D.J., O’Donnell L., Jamsai D., Bergmann M, & O’Bryan M.K. (2017). Katanin-like 2 (KATNAL2) functions in multiple aspects of haploid male germ cell development in the mouse. PLoS Genetics, 13(11), e1007078.

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Placental Protein Expression Analysis

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Western blot (WB) was performed in 4 PE samples and 4 control samples using placental tissues. Briefly, protein was extracted from placental samples by the methods of TRIzol. Block the membrane with 5% skim milk in Tris-Buffered Saline Tween-20 (TBST) for 1 h. Primary antibody was used in 5% bovine serum albumin (BSA) and incubated at room temperature for 2 h, then washed the membrane with TBST for 4 times, 8 min each time. The secondary antibody was incubated for 1.5 h, and the membrane was washed with TBST for 3 times. Membrane blots were visualized using ECL plus detection kit (Thermo Fisher Scientific) and imaged on ImageQuant 4600 (Tenon, Shanghai, China).

Wang J., Gao F., Zhao X., Cai Y, & Jin H. (2020). Integrated analysis of the transcriptome-wide m6A methylome in preeclampsia and healthy control placentas. PeerJ, 8, e9880.

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Measuring Antioxidant and Apoptotic Markers in Myocardial Tissue

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The enzymatic activity of superoxide dismutase (SOD) was measured by a SOD assay kit (Sigma, Switzerland) and glutathione content was measured by GSH-Glo Glutathione assay kit (Promega, Madison, WI) according to protocols provided by the companies. A 20/20 luminometer (Turner BioSystems, Sunnyvale, CA) was used to detect total glutathione levels while a Spectra Max Plus (Sunnyvale, CA) was used to detect the total SOD activity. Myocardial protein content of cleaved caspase-3 (Santa Cruz Biotechnology), hypoxia-inducible factor 1alpha (Hif-1α) (Novus Biologicals, Littleton, CO) from LV homogenates were determined by Western blot analysis as described previously (Zhang et al. 2007 (link)). Briefly, total protein from LV tissue was extracted by T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL). Protein samples (25–30 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. The membranes were probed with corresponding primary antibodies. Appropriate HRP-conjugated secondary antibodies were used and the antibody-antigen complexes in all membranes were detected by the ECL PLUS Detection Kit (Thermo Scientific). The expression of these proteins was quantified with Scion Image (NIH, Bethesda, MD) and adjusted to β-actin.

Gupta P.K., DiPette D.J, & Supowit S.C. (2014). Protective effect of resveratrol against pressure overload-induced heart failure. Food Science & Nutrition, 2(3), 218-229.

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