Ecl plus western detection kit

Manufactured by GE Healthcare

The ECL Plus Western detection kit is a chemiluminescent detection system used in Western blotting analysis to detect and quantify proteins. The kit provides reagents for enhanced chemiluminescent (ECL) detection of horseradish peroxidase (HRP)-labeled proteins on membranes.

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Lab products found in correlation

Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions) Anti actin, by Merck Group (1 mentions) P9599, by Merck Group (1 mentions) Image quant las 4000 mini ccd camera system, by GE Healthcare (1 mentions) Anti ha 11, by BioLegend (1 mentions) Las 4000, by Cytiva (1 mentions) Goat anti rabbit hrp, by Agilent Technologies (1 mentions) Goat anti mouse hrp, by Agilent Technologies (1 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Hyperfilm mp, by GE Healthcare (1 mentions) Proteinase inhibitor cocktail, by Roche (1 mentions) Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)

Spelling variants of this product

ECL Plus (592 citations) ECL kit (450 citations) ECL detection kit (236 citations) ECL Plus kit (158 citations) ECL Plus Western (2 citations)

3 protocols using ecl plus western detection kit

Protein Extraction and Western Blot

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Proteins were extracted in 50 mM Na-phosphate (pH 7.4), 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50 µM MG132, 2 mM Na₃VO₄, 2 mM NaF, and 1% (v/v) protease inhibitor mixture for plant extracts (Sigma-Aldrich, P9599). For immunoblot analysis, total cellular proteins were separated by electrophoresis in 10% (w/v) SDS–polyacrylamide gels and transferred to PVDF membranes according to the manufacturer's instructions (iBlot dry blotting system, Thermo Fisher Scientific).
Rabbit polyclonal antibodies were generated against synthetic peptides derived from the RUP2 protein sequence [amino acids 1–15 + C: MNTLHPHKQQQEQAQC; anti-RUP2^(1–15)] and were affinity-purified against the peptide (Eurogentec). Anti-RUP2^(1–15), anti-UVR8^(426–440) (Favory et al. 2009 (link)), anti-HA.11 (BioLegend, 901513), and anti-actin (Sigma-Aldrich, A0480) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse immunoglobulins (Dako A/S) were used as the secondary antibodies. Chemiluminescent signals were generated with the ECL Plus Western detection kit and revealed with an ImageQuant LAS 4000 mini-CCD camera system (GE Healthcare).

Arongaus A.B., Chen S., Pireyre M., Glöckner N., Galvão V.C., Albert A., Winkler J.B., Fankhauser C., Harter K, & Ulm R. (2018). Arabidopsis RUP2 represses UVR8-mediated flowering in noninductive photoperiods. Genes & Development, 32(19-20), 1332-1343.

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Western Blot Analysis of UHRF1 Protein

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Forty-eight hours after transfection, cells were harvested with ice-cold lysis buffer (0.2 M KCl, 0.03 M Tris, pH 7.25) containing proteinase inhibitor cocktail (Roche Diagnostics). Lysates were boiled in equal volumes of loading buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% ß-mercaptoethanol), and 20 μg of protein was separated in a 8–12% gradient Tris-glycine gradient gel (Novex, San Diego, CA, USA) under reducing conditions and transferred to nitrocellulose membranes (GE Healthcare, Piscataway, USA). Afterwards, membranes were blocked at room temperature with PBS containing 0.1% Tween and 5% non-fat dry milk to block non-specific binding for 2 h. Membranes were incubated with primary antibodies (rabbit anti-human ß-actin, Cell Signaling Technology, Danvers, USA; mouse anti-human UHRF1, generously provided by Dr. C. Bronner, Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France) at 4 °C overnight, followed by a 1-h incubation with the corresponding secondary antibodies (goat anti-rabbit-HRP and goat anti-mouse-HRP, both Dako Cytomation, Hamburg, Germany) at room temperature. For chemiluminescent detection, the ECL Plus Western detection kit (GE Healthcare) was used. Protein bands were detected by autoradiography using the high-performance autoradiography Hyperfilm™ MP (GE Healthcare).

Beck A., Trippel F., Wagner A., Joppien S., Felle M., Vokuhl C., Schwarzmayr T., Strom T.M., von Schweinitz D., Längst G, & Kappler R. (2018). Overexpression of UHRF1 promotes silencing of tumor suppressor genes and predicts outcome in hepatoblastoma. Clinical Epigenetics, 10, 27.

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Western Blotting Analysis Protocol

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For western blotting analysis, the native-PAGE and SDS–PAGE gels were prepared according to a previously described method (Laemmli 1970 (link)), and were then stained with Quick Coomassie brilliant blue (CBB) (Wako, Osaka, Japan) or transferred into polyvinylidene fluoride (PVDF) membranes (Roch) under 1.0 mA for 30 min. The PVDF membrane was then blocked in 3% Tris-buffered saline and Tween 20 (TBST) skim milk for 1 h at 4°C, followed by incubation with anti-BmAOX5 primary antibody (1:2,000, synthesized using an immunized rabbit treated with BmAOX5-special polypeptides [amino acid positions 33–45, RAKRYPSGKTETF], Genscript Nanjing Co. Ltd.) in 3% TBST skim milk for 1 h at room temperature (RT, about 25°C). The membrane was washed 5× with TBST for 5 min in a rotary shaker and incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Sigma–Aldrich, St. Louis, MO) in 3% TBST skim milk for 1 h at RT. The membrane was washed 3× with TBST for 10 min in a rotary shaker and then incubated with the chemiluminescent substrate from the ECL Plus Western Detection Kit (GE Healthcare, Chicago, IL) for 5 min and photographed using a Clinx ChemiScope 3400 Mini (Science Instrument, Beijing, China).

Zhang Y., Yang Y., Shen G., Mao X., Jiao M, & Lin Y. (2020). Identification and Characterization of Aldehyde Oxidase 5 in the Pheromone Gland of the Silkworm (Lepidoptera: Bombycidae). Journal of Insect Science, 20(6), 31.

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