Western lightning ecl reagent

Manufactured by PerkinElmer

Sourced in United States

The Western Lightning ECL reagent is a chemiluminescent detection system used for the visualization of proteins in Western blot analysis. The reagent contains a proprietary mixture of chemicals that produce a light-emitting reaction when combined with the target proteins, allowing for the detection and quantification of specific proteins in a sample.

Automatically generated - may contain errors

Lab products found in correlation

Gel doc chemi doc imaging system, by Bio-Rad (3 mentions) Laemmli buffer, by Bio-Rad (3 mentions) Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions) Protease inhibitor, by Roche (3 mentions) Imagej software, by Thermo Fisher Scientific (2 mentions) Bradford assay, by Bio-Rad (2 mentions) Tristar lb 941 luminometer, by Berthold Technologies (2 mentions) Maxisorp nunc plates, by Thermo Fisher Scientific (2 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Alpha imager, by Bio-Techne (1 mentions) Total gsk3β, by Cell Signaling Technology (1 mentions) β catenin, by Merck Group (1 mentions) P smad3, by Abcam (1 mentions) P smad2, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Phospho erk t202 y204, by Cell Signaling Technology (1 mentions) Phospho akt s473, by Cell Signaling Technology (1 mentions) Phospho rtk array, by R&D Systems (1 mentions) Phospho s6 s240 244, by Cell Signaling Technology (1 mentions) P smad2, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

ECL reagent (117 citations) Western Lightning (69 citations) Western Lightning ECL (49 citations) Western Lightning reagent (17 citations) Western Lightning Chemiluminescence (ECL) reagents (2 citations)

20 protocols using western lightning ecl reagent

Western Blot Analysis of Sporulating B. subtilis

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Whole-cell lysates from sporulating cells were prepared as previously described (Doan and Rudner, 2007 (link)). Samples were heated for 5 min at 65°C prior to loading. Equivalent loading of proteins was based on the OD₆₀₀ of the cell cultures at the time of harvest. Samples were separated on a 12.5% SDS-polyacrylamide gel and transferred to a methanol-activated PVDF membrane. Membranes were blocked in 5% nonfat milk with 0.5% Tween-20 for 1 hour. Blocked membranes were probed with anti-σ^H (diluted 1:2,500), anti-Spo0A (diluted 1:5,000) or anti-SpoIIQ (diluted 1:10,000) (Doan et al., 2009 (link)), anti-σ^A (diluted 1:10,000), anti-KinA (diluted 1:5,000), and anti-core RNA-polymerase (diluted 1:5,000). These primary antibodies were diluted into PBS with 0.05% Tween-20 and incubations were carried out at 4°C overnight. Primary antibodies were detected with horseradish-peroxidase conjugated anti-mouse or anti-rabbit antibodies and detected with Western Lightning ECL reagent as described by the manufacturer (PerkinElmer, Waltham, MA, USA).

Widderich N., Rodrigues C.D., Commichau F.M., Fischer K.E., Ramirez-Guadiana F.H., Rudner D.Z, & Bremer E. (2016). Salt-sensitivity of σH and Spo0A prevents sporulation of Bacillus subtilis at high osmolarity avoiding death during cellular differentiation. Molecular microbiology, 100(1), 108-124.

+ Open protocol

+ Expand

Western Blot Protein Detection Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

SDS-PAGE gels including Precision Plus Stained protein standards (Thermo Fisher) were transferred to activated 0.2 μm PVDF membrane (Thermo Scientific) via semi-dry transfer in (25 mM Tris-HCl pH 8.3, 192 mM glycine, 5% methanol) for 45 min using constant 80 mA current before membrane blocking with 5% milk in TBST (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20) for at least one hour. Blocked membranes were probed with primary antibody (diluted in TBST with 5% milk) for at least 1 hr before being washed with TBST and re-probed with secondary anti-Rabbit-HRP for at least 30 min. Membranes were subsequently washed three times with TBST (15 min each) before visualization of Chemiluminescence activity using Western Lightning ECL reagent (Perkin Elmer) in Chemidoc MP Imaging System (Bio-Rad) with exposure times ranging from 30 to 120 s.

Greene E.R., Goodall E.A., de la Peña A.H., Matyskiela M.E., Lander G.C, & Martin A. (2019). Specific lid-base contacts in the 26s proteasome control the conformational switching required for substrate degradation. eLife, 8, e49806.

+ Open protocol

+ Expand

Assessing CaMKIIα Autophosphorylation

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

To assess the ability of CaMKIIα WT and mutants to autophosphorylate at T286, the kinases were added at a final concentration of 20 nM to a reaction buffer containing 50 mM PIPES pH 7.2, 0.1% BSA, 2 mM CaCl₂, 10 mM MgCl₂, 100 mM ATP, 1 μM microcystin-LR, 100 nM calmodulin. Kinases were reacted at 30 °C for 30 seconds, or as indicated. Reactions were stopped by addition of gel loading buffer (2% SDS, 50 mM dithiothreitol, 67.5 mM Tris pH 6.8, 10% glycerol, 0.16 mg ml⁻¹ bromophenol blue) and boiling for 5 min. Samples were loaded on 10% SDS–polyacrylamide gel electrophoresis gels then transferred to polyvinylidene difluoride membranes42 (link)43 . Blots were blocked in 5% milk then probed with anti-phospho-T286 CaMKII antibody (Phosphosoutions) diluted 1:3,000 in 1% milk. Images were acquired on an Alpha Imager (Alpha Innotech) after exposure to Western Lightning ECL reagent (Perkin Elmer) and quantified42 (link)43 44 (link).

Myers J.B., Zaegel V., Coultrap S.J., Miller A.P., Bayer K.U, & Reichow S.L. (2017). The CaMKII holoenzyme structure in activation-competent conformations. Nature Communications, 8, 15742.

+ Open protocol

+ Expand

Muscle Progenitor Cell Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human or mouse primary muscle progenitor cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and 1 mM EDTA, pH 7.4) containing 1X protease inhibitor (Roche), 1 mM Phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride and 1 mM sodium orthovanadate. Cell lysates were resuspended in 1X Laemmli buffer (Bio-Rad), boiled for 5 minutes and separated on precast 7.5% or 4-15% TGX gels (Biorad). Primary antibodies were diluted in 5% non-fat milk in TBS + 0.1% Tween-20, and nitrocellulose membranes were incubated with antibody mixtures overnight at 4 °C. HRP-conjugated secondary antibodies (Santa Cruz Biotech) were diluted 1:1,000 in 5% non-fat milk in TBS + 0.1% Tween-20 and incubated for 1 hour at room temperature. Blots were developed using Western Lightning ECL reagent (Perkin Elmer), and analyzed with Bio-Rad Gel Doc/Chemi Doc Imaging System and Quantity One software. Antibodies for phospho-ERK1/2, ERK1/2, pSmad1,5,8 and β-Actin, pGSK3β Y216, and Total GSK3β, were purchased from Cell Signaling. pSmad3 was purchased from Epitomics. Smad2/3, and Dll1 antibodies were from Santa Cruz Biotechnology. GapDH and Nicd1 antibodies were from Abcam, pSmad2 and β-Catenin were from Millipore.

Yousef H., Conboy M.J., Mamiya H., Zeiderman M., Schlesinger C., Schaffer D.V, & Conboy I.M. (2014). Mechanisms of action of hESC-secreted proteins that enhance human and mouse myogenesis. Aging (Albany NY), 6(8), 602-620.

+ Open protocol

+ Expand

EGFR Signaling Pathway Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed with PBS and lysed in radioimmunoprecipitation analysis buffer (50 mM TrisHCl pH 8.0, 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton X-100. 0.5% sodium deoxycholate, 0.1% SDS, 40 mM sodium fluoride, 1 mM sodium orthovanadate, and complete Protease Inhibitor Cocktail [Roche Diagnostics, Indianapolis, IN, USA]). Western Lightning ECL reagent (PerkinElmer, Waltham, MA, USA) was used for signal detection. β-actin antibody (A2066) was purchased from Sigma-Aldrich. EGFR (#2232), pEGFR Y1068 (#2234), pEGFR Y1173 (#2244), ERK (#9102), pERK T202/Y204 (#9101), horseradish peroxidase (HRP)-conjugated anti-mouse (#7076) and HRP-conjugated anti-rabbit (#7074) antibodies were purchased from Cell Signaling (Danvers, MA, USA). Each experiment was performed twice.

Brown B.P., Zhang Y.K., Westover D., Yan Y., Qiao H., Huang V., Du Z., Smith J.A., Ross J.S., Miller V.A., Ali S., Bazhenova L., Schrock A.B., Meiler J, & Lovly C.M. (2019). On-target resistance to the mutant-selective EGFR inhibitor osimertinib can develop in an allele specific manner dependent on the original EGFR activating mutation. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(11), 3341-3351.

+ Open protocol

+ Expand

Immunoblotting Analysis of Cellular Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Resistant cells were removed from drug selection 72 hours before immunoblotting experiments. Cells were washed on ice with cold PBS and lysed in radioimmunoprecipitation (RIPA) buffer (250mM Tris-HCl pH 7.5, 75mM NaCl, 1% NP-40/IGEPAL, 0.1% sodium dodecyl sulfate) supplemented with complete protease inhibitor cocktail (Roche), 40mM sodium fluoride, 1mM sodium orthovanadate, and 1uM okadaic acid. Lysates were subjected to SDS-PAGE (4-12% gels) followed by immunoblotting with the indicated antibodies and detection by Western Lightning ECL reagent (Perkin-Elmer). The following antibodies were obtained from Cell Signaling Technologies: phospho-ERK (T202/Y204; 1:1000; # 9101), ERK (1:1000, #9102), phospho-AKT (S473; 1:500; #9271), AKT (1:1000; #9272), phospho-S6 (S240/244; 1:1000; #2215), S6 (1:1000; #2217), BIM (1:1000; #2819), HRP-conjugated anti-mouse (1:3000; #7076), and HRP-conjugated anti-rabbit (1:3000; #7074). Phospho-EGFR antibody was obtained from Abcam (Y1068; 1:1000; #EP774Y), EGFR from BD Transduction Laboratories (1:500; #610017), and actin from Sigma-Aldrich (1:3000; #A2066). Phospho-RTK arrays were purchased from R&D Systems (#ARY001B), and assays were run on cells maintained in 10% FBS using the manufacturer’s protocol.

Meador C.B., Jin H., de Stanchina E., Nebhan C.A., Pirazzoli V., Wang L., Lu P., Vuong H., Hutchinson K.E., Jia P., Chen X., Eisenberg R., Ladanyi M., Politi K., Zhao Z., Lovly C.M., Cross D.A, & Pao W. (2014). Optimizing the sequence of anti-EGFR targeted therapy in EGFR-mutant lung cancer. Molecular cancer therapeutics, 14(2), 542-552.

+ Open protocol

+ Expand

Cell Lysis and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed using RIPA buffer (0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mM Tris pH 7.4) plus protease and phosphatase inhibitors (cOmplete Mini, EDTA-free and PhosphoSTOP; Roche, Indianapolis, IN). Following SDS-PAGE, protein was transferred to nitrocellulose, blocked and incubated with primary antibodies [MMP2 (Abcam), pSmad 2 (Cell Signaling), or Actin (Sigma)]. Secondary antibodies were directly HRP-conjugated (Cell Signaling) or biotinylated (Vector, Burlingame, CA) and detected with streptavidin-HRP. Chemiluminescent detection was achieved using Western Lightning ECL reagent (PerkinElmer, Waltham, MA).

Bates A.L., Pickup M.W., Hallett M.A., Dozier E.A., Thomas S, & Fingleton B. (2015). Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases. The Journal of pathology, 235(5), 773-783.

+ Open protocol

+ Expand

Western Blot Analysis of Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) : EGFR (#2232), pEGFR Y1068 (#2234), HER3 (#4754), pHER3 Y1197 (#4561), pBRAF S445 (#2696), MEK (#9126), pMEK S217/221 (#9154), ERK (#9102), pERK T202/Y204 (#9101), PDK1 (#3062), pPDK1 S241 (#3061), AKT (#9272), pAKT S473 (#9271), pAKT T308 (#9275), ribosomal protein S6 (#2317), pS6 S240/S244 (#5364), FAK (#3285), pFAK Y397 (#8556), SFKs (#2108), pSFKs Y416 (#2101), YES (#3201), Integrin Antibody Sampler Kit (#4749), PARP (#9542), E-cadherin (#3195), Vimentin (#3932), horseradish peroxidase (HRP)-conjugated anti-mouse (#7076) and HRP-conjugated anti-rabbit (#7074). The actin antibody (A2066) was purchased from Sigma-Aldrich. The BRAF antibody (sc-55522) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). For immunoblot, cells were harvested, washed in PBS, and lysed in RIPA buffer [50 mM Tris•HCl (pH 8.0), 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton X-100. 0.5% sodium deoxycholate, 0.1% SDS, 40 mM sodium fluoride, 1 mM sodium orthovanadate, and complete protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA)]. Western Lightning ECL reagent (Perkin-Elmer) as used for signal detection. Phosphorylated bands were quantified using ImageJ software.

Ichihara E., Westover D., Meador C.B., Yan Y., Bauer J.A., Lu P., Ye F., Kulick A., de Stanchina E., McEwen R., Ladanyi M., Cross D., Pao W, & Lovly C.M. (2017). SFK/FAK signaling attenuates osimertinib efficacy in both drug-sensitive and drug-resistant models of EGFR-mutant lung cancer. Cancer research, 77(11), 2990-3000.

+ Open protocol

+ Expand

Dot Blot Assay for 5-hmC Detection

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

Dot blot assays were performed as previously described19 (link). Briefly, genomic DNA was sonicated, 5-hmC was conjugated to UDP-6-N3-Glu18 (link), and biotinylated by addition of 4.5 nm Click-IT Biotin DIBO Alkyne (Life Technologies). The biotin labeled DNA was purified and spotted on HyBond N + membrane (GE Healthcare Life Sciences). Membranes were probed with Avidin-HRP and dots were visualized with Western Lightning ECL reagent (PerkinElmer).

Chapman C.G., Mariani C.J., Wu F., Meckel K., Butun F., Chuang A., Madzo J., Bissonnette M.B., Kwon J.H, & Godley L.A. (2015). TET-catalyzed 5-hydroxymethylcytosine regulates gene expression in differentiating colonocytes and colon cancer. Scientific Reports, 5, 17568.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primary neural or muscle progenitor cells, or whole muscle tissue were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and 1 mM EDTA, pH 7.4) containing 1X protease inhibitor (Roche), 1 mM Phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium fluoride and 1 mM sodium orthovanadate. The protein concentration was determined by Bradford assay (Bio-Rad). Lysates were resuspended in 1X Laemmli buffer (Bio-Rad), boiled for 5 minutes and separated on precast 7.5% or 4–15% TGX gels (Biorad). Primary antibodies were diluted in 5% non-fat milk in TBS + 0.1% Tween-20, and nitrocellulose membranes were incubated with antibody mixtures overnight at 4°C. HRP-conjugated secondary antibodies (Santa Cruz Biotech) were diluted 1:500 in 5% non-fat milk in TBS + 0.1% Tween-20 and incubated for 1 hour at room temperature. Blots were developed using Western Lightning ECL reagent (Perkin Elmer), and analyzed with Bio-Rad Gel Doc/Chemi Doc Imaging System and Quantity One software. Results of multiple assays were quantified using Applied Biosystems or Image J software. Pixel Intensity of bands of interest were normalized with pixel intensity of glyceraldehydes-3-phosphate dehydrogenase or β-actin.

Yousef H., Conboy M.J., Morgenthaler A., Schlesinger C., Bugaj L., Paliwal P., Greer C., Conboy I.M, & Schaffer D. (2015). Systemic attenuation of the TGF-β pathway by a single drug simultaneously rejuvenates hippocampal neurogenesis and myogenesis in the same old mammal. Oncotarget, 6(14), 11959-11978.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!