Western lightning ecl kit

Manufactured by PerkinElmer

Sourced in United States

The Western Lightning ECL Kit is a chemiluminescent detection system used for the analysis of proteins in Western blotting applications. It provides a sensitive and reliable method for the detection of target proteins.

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Lab products found in correlation

Nitrocellulose membrane, by Bio-Rad (4 mentions) β actin, by Merck Group (3 mentions) Monoclonal rat anti mouse 1c7, by Amgen (2 mentions) α actin, by Merck Group (1 mentions) Epitect fast bisulfite kit, by Qiagen (1 mentions) α h3k4me3, by Merck Group (1 mentions) Qiaamp dna mini kit, by Qiagen (1 mentions) Hybond c membrane, by Cytiva (1 mentions) Criterion pre cast gradient gels, by Bio-Rad (1 mentions) Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions) Las 3000 intelligent dark box, by Fujifilm (1 mentions) Ab291, by Abcam (1 mentions) Pi 2000, by Vector Laboratories (1 mentions) Chemidoc touch imager, by Bio-Rad (1 mentions) Sc 9000, by Santa Cruz Biotechnology (1 mentions) Sc 47724, by Santa Cruz Biotechnology (1 mentions) Precellys 24, by Avantor (1 mentions) Lowry assay, by Bio-Rad (1 mentions) Nitrocellulose filter, by Bio-Rad (1 mentions) Phospho smad1 5 8, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Western Lightning Plus-ECL chemiluminescence kit Western Lightning Plus-ECL reagent kit Western Lightning Plus-ECL chemiluminescence detection kit Western Lightning Plus-ECL enhanced chemiluminescence detection kit Western Lightning Plus-ECL kit Western Lightning Plus ECL detection kit Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate kit Western Lightning ECL Pro kit Western Lightning ECL Pro development kit Western Lightning enhanced chemiluminescence kit (ECL)

11 protocols using western lightning ecl kit

Profiling Hippocampal Histone Marks

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Western blot analyses were carried out using Western Lightning ECL kit (Perkin-Elmer, Boston, MA), and α-H3K4me3 (Millipore 07-473) and α-actin (Sigma F5441) antibodies. DNA was extracted from adult hippocampi with QIAamp DNA Mini Kit (Qiagen). Bisulfite conversion of genomic DNA was performed with the EpiTect Fast bisulfite kit (Qiagen). Primer sequences for PCR amplification and pyrosequencing are provided in Supplemental Experimental Procedures.

Scandaglia M., Lopez-Atalaya J.P., Medrano-Fernandez A., Lopez-Cascales M.T., del Blanco B., Lipinski M., Benito E., Olivares R., Iwase S., Shi Y, & Barco A. (2017). Loss of Kdm5c causes spurious transcription and prevents the fine-tuning of activity-regulated enhancers in neurons. Cell reports, 21(1), 47-59.

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Western Blot Analysis of Proteins

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Proteins were resolved by SDS-PAGE on Criterion Precast gradient gels (4-15%, Biorad). and transferred to Hybond-C membrane (Amersham Biosciences). The membrane was incubated overnight at 4°C with either: (i) affinity purified anti-EMB-4, or anti alpha-tubulin (Accurate Chemical) antibodies diluted to 1:2000, in PBST-5% milk solution (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4, and 5% [w/v] dried milk). The membrane was incubated 1 h at room temperature with HRP-conjugated secondary antibodies (Jackson Immunoresearch) diluted to 1:5,000 in PBST and then visualized by Western Lightning ECL Kit from Perkin Elmer. Images were collected on a LAS-3000 Intelligent Dark-Box (Fujifilm).

Akay A., Di Domenico T., Suen K.M., Nabih A., Parada G.E., Larance M., Medhi R., Berkyurek A.C., Zhang X., Wedeles C.J., Rudolph K.L., Engelhardt J., Hemberg M., Ma P., Lamond A.I., Claycomb J.M, & Miska E.A. (2017). The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription. Developmental Cell, 42(3), 241-255.e6.

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Density-based Protein Separation and Analysis

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Densities of the 1-ml fractions from the flotation centrifugation analyses were determined from three independent experiments by measuring the weight of three 100 µl subfractions from each fraction. The fractions were precipitated with trichloroacetic acid (TCA) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gels were stained with Coomassie, Sudan black, or both and imaged using a ChemiDoc Touch imager (BioRad) or used for Western blotting. Polyclonal antibodies (pAb) against P12 (1:13,333; GeneCust) and phi6 (1:2500) and a monoclonal antibody against GFP (1:25,000; ab291, Abcam) were used as primary antibodies. HRP-labeled anti-rabbit pAb (1:125,000; Sigma Aldrich) or anti-mouse pAb (1:5000; PI-2000; Vector Laboratories Inc.) were used as secondary antibodies. Chemiluminescence was detected using a Western Lightning ECL Kit (Perkin-Elmer) and ChemiDoc Touch imager (BioRad) with 600 s of exposure time. Mass spectrometric analyses of P12 were performed in the Proteomics Unit of the University of Helsinki using liquid-chromatography–mass spectrometry × 2 (LC–MS/MS). Quantitative analyses of the EM thin sections was done using Aida Image Analyzer v 4.5.

Lyytinen O.L., Starkova D, & Poranen M.M. (2019). Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6. Microbial Cell Factories, 18, 29.

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Western Blot Analysis of Ferroportin

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Spleens were washed with ice-cold PBS and dissected into pieces. Aliquots were snap-frozen in liquid nitrogen and stored at −80 °C. Protein lysates were obtained as described [42 (link)]. The lysates containing 40 μg of proteins were analyzed using SDS–PAGE on 9–13% gels, and the proteins were transferred onto nitrocellulose membranes (BioRad). The blots were blocked in non-fat milk diluted in tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 (TBS-T) and probed overnight with antibodies against ferroportin [71 (link)] (1:1000 diluted monoclonal rat anti-mouse 1C7, kindly provided by Amgen Inc), β-actin (1:2000 diluted; Sigma). Following a 3× wash with TBS-T, the membranes were incubated with peroxidase-coupled secondary antibodies for 1.5 h. Immunoreactive bands were detected via enhanced chemiluminescence with a Western Lightning ECL Kit (Perkin Elmer, Waltham, MA, USA). Blot images were quantified using ImageJ software (version 1.53t).

Charlebois E., Li Y., Wagner V., Pantopoulos K, & Olivier M. (2023). Genetic Iron Overload Hampers Development of Cutaneous Leishmaniasis in Mice. International Journal of Molecular Sciences, 24(2), 1669.

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Western Blot Analysis of Iron Regulators

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Livers were washed with ice-cold PBS and dissected into pieces. Aliquots were snap frozen at liquid nitrogen and stored at −80°C. Protein lysates were obtained as described (Katsarou et al., 2021 (link)). Lysates containing 40 μg of proteins were analyzed by SDS-PAGE on 9–13% gels and proteins were transferred onto nitrocellulose membranes (BioRad). The blots were blocked in non-fat milk diluted in tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 (TBS-T), and probed overnight with antibodies against ferroportin (Ross et al., 2017 (link); 1:1000 diluted monoclonal rat anti-mouse 1C7, kindly provided by Amgen Inc), β-actin (1:2000 diluted; Sigma), Tfr2 (1:1000 diluted rabbit polyclonal; Alpha Diagnostics), or Tfr1 (1:1000 diluted mouse monoclonal, Invitrogen). Following a 3 × wash with TBS-T, the membranes were incubated with peroxidase-coupled secondary antibodies for 1 hr. Immunoreactive bands were detected by enhanced chemiluminescence with the Western Lightning ECL Kit (Perkin Elmer).

Charlebois E., Fillebeen C., Katsarou A., Rabinovich A., Wisniewski K., Venkataramani V., Michalke B., Velentza A, & Pantopoulos K. (2022). A crosstalk between hepcidin and IRE/IRP pathways controls ferroportin expression and determines serum iron levels in mice. eLife, 11, e81332.

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Western Blot Analysis of PPARα and CrAT

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PPARα protein levels from nuclear extracts were analysed by Western blot. Nuclear extracts were prepared after an established protocol as described above. Aliquots containing 25 μg of nuclear extract were loaded on to a 10% SDS polyacrylamid gel. After electrophoresis proteins were transferred to a nitrocellulose membrane. The membrane was probed with antibodies against PPARα (sc-9000, Santa Cruz Biotechnology) and GAPDH (sc-47724, Santa Cruz Biotechnology). In the case of the CrAT western blot 10 μg whole cell protein extracts were separated on 12% SDS polyacrylamid gels and probed with CrAT antibody (Abcam ab91478) and GAPDH (sc-47724, Santa Cruz Biotechnology). The membranes were then processed with HRP conjugated secondary antibodies specific for the appropriate species. Proteins were visualized with Western Lightning ECL kit (Perkin Elmer). The statistical analyses were carried by SPSS software (IBM) using the t-test subroutine for independent samples (student’s T test) to calculate the p-values given in the figure legends. In the case of Figure 4 we performed a Kruskal-Wallis test with GraphPad Prism (GraphPad Software Inc.).

Kienesberger K., Pordes A.G., Völk T.G, & Hofbauer R. (2014). L-carnitine and PPARα-agonist fenofibrate are involved in the regulation of Carnitine Acetyltransferase (CrAT) mRNA levels in murine liver cells. BMC Genomics, 15(1), 514.

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Sciatic Nerve Protein Analysis

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Sciatic nerves were transferred to 200 µl (per nerve) ice-cold lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, and protease inhibitors (Complete, Roche, Switzerland). Samples were homogenized using Precellys 24 (VWR International, Dealware, USA), centrifuged for 10 min at 1000 g to remove cellular nuclei, and protein concentration of the supernatant was determined using Lowry assay (Bio-rad Laboratories, California, USA). Blotting membranes were incubated overnight at 4°C with primary antibodies. Western Lightning + ECL Kit (Perkin Elmer, Massachusetts, USA) was used to label protein bands, which were detected using Intas ChemoCam Image system. At least four biological replicates were analyzed. Quantification was performed in Fiji.

Kleinecke S., Richert S., de Hoz L., Brügger B., Kungl T., Asadollahi E., Quintes S., Blanz J., McGonigal R., Naseri K., Sereda M.W., Sachsenheimer T., Lüchtenborg C., Möbius W., Willison H., Baes M., Nave K.A, & Kassmann C.M. (2017). Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy. eLife, 6, e23332.

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Western Blot Analysis of Phospho-Smad1/5/8

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Liver lysates were prepared as described earlier [17] (link). All samples (containing 30 µg of protein) were analyzed by SDS-PAGE on 10% gels by standard procedures. Following transfer of the proteins onto nitrocellulose filters (BioRad), the blots were saturated with 5% non-fat milk in phosphate buffered saline (PBS) containing 0.1% (v/v) Tween-20 (PBS-T) for 1 hour, and probed overnight with a 1∶1000 diluted antibodies against phospho-Smad1/5/8 or Smad1 (Cell Signaling). After three washes with PBS-T, the blots were incubated with 1∶25000 diluted peroxidase-coupled goat anti-rabbit IgG (Sigma) for 1 hour. The peroxidase signal was detected by enhanced chemiluminescence with the Western Lightning ECL kit (Perkin Elmer) and quantified by densitometry.

Gkouvatsos K., Fillebeen C., Daba A., Wagner J., Sebastiani G, & Pantopoulos K. (2014). Iron-Dependent Regulation of Hepcidin in Hjv−/− Mice: Evidence That Hemojuvelin Is Dispensable for Sensing Body Iron Levels. PLoS ONE, 9(1), e85530.

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Western Blot Quantification Workflow

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Proteins (5 μg) were separated by SDS–polyacrylamide gel electrophoresis on 4–20% Mini-Protean TGX gels (cat#4561096; Bio-Rad Laboratories; Hercules, CA) and were blotted to polyvinylidene difluoride membranes. Blots were blocked with 3% (w/v) non-fat milk in phosphate-buffered saline solution containing 0.05% (v/v) Tween 20 (PBS-T) and then incubated overnight at 4°C with the primary antibodies listed in Table 2. Membranes were rinsed three times in PBS-T and were incubated for 1 h with the corresponding horseradish peroxidase-conjugated secondary antibodies (Table 2). Blots were developed by enhanced chemiluminescence using the Western Lightning ECL™ kit (cat#NEL103; Perkin-Elmer, Boston, MA) and signals captured on blue X-ray films. Films were scanned in a Hewlett Packard Scanjet 4050 and the images were quantified using the NIH Fiji ImageJ 1.52 imaging analysis program (RRID:SCR_003070). Band intensities were normalized by the intensity of the Coomassie blue stain in the respective lanes or, in the case of the nuclear fractions, by the amount of histone H3. All the antibodies used in this study were validated by the suppliers and detected bands at the predicted molecular weights on western blots.

Shanley K.L., Hu C.L, & Bizzozero O.A. (2019). Decreased levels of constitutive proteasomes in experimental autoimmune encephalomyelitis may be caused by a combination of subunit displacement and reduced Nfe2l1 expression. Journal of neurochemistry, 152(5), 585-601.

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Immunoblotting Analysis of Phosphorylated Signaling Proteins

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Following 2x wash with phosphate-buffered-saline, the cells were lysed as previously described [21 (link)]. Cell lysates containing 30 μg of proteins were analyzed by SDS-PAGE on 10% gels and proteins were transferred onto nitrocellulose membranes (BioRad). The blots were blocked in 10% bovine serum albumin or 10% fat-free skim milk in tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 (TBS-T), and probed overnight with 1:1000-diluted (unless otherwise indicated) primary antibodies against pSMAD5 (phospho-S463/465, #92698 rabbit monoclonal; Abcam), SMAD1 (#9743 rabbit polyclonal; Cell Signaling), p STAT3 (phospho-Y705, #9138, mouse monoclonal; Cell Signaling), STAT3 (#9139, mouse monoclonal; Cell Signaling), ferritin heavy chain (1:500-diluted, #NB600-920, Novus), or β-actin (Sigma). Following a 3x wash with TBS-T, the membranes were incubated with 1:5000-diluted anti-mouse or 1:20000-diluted anti-rabbit peroxidase-coupled secondary antibodies for 2 h. Immunoreactive bands were detected by enhanced chemiluminescence with the Western Lightning ECL Kit (Perkin Elmer). Original Western blot images can be found in S1 Raw images.

Charlebois E, & Pantopoulos K. (2021). Iron overload inhibits BMP/SMAD and IL-6/STAT3 signaling to hepcidin in cultured hepatocytes. PLoS ONE, 16(6), e0253475.

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