Western lightning

Manufactured by PerkinElmer

Sourced in United States

Western Lightning is a chemiluminescent detection reagent used in Western blot analysis. It is designed to detect and quantify proteins that have been separated by gel electrophoresis and transferred to a membrane.

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Lab products found in correlation

Las 3000 imaging system, by Fujifilm (6 mentions) Nupage, by Thermo Fisher Scientific (6 mentions) Immobilon p, by Merck Group (5 mentions) Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (5 mentions) Pvdf membrane, by Merck Group (5 mentions) Bca protein assay kit, by Beyotime (4 mentions) Fluorchem digital imager, by Bio-Techne (3 mentions) Immobilon, by Merck Group (3 mentions) β actin, by Santa Cruz Biotechnology (3 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions) Protease and phosphatase inhibitor cocktail, by Roche (3 mentions) Nitrocellulose membrane, by GE Healthcare (3 mentions) Radioimmunoprecipitation assay (ripa), by Boston BioProducts (2 mentions) Tgx 4 20 gel, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Protein assay kit, by Bio-Rad (2 mentions) Anti actin, by Merck Group (2 mentions) Sds gel loading buffer, by Thermo Fisher Scientific (2 mentions) Hiv ig serum, by Jackson ImmunoResearch (2 mentions) Bis tris gel, by Lonza (2 mentions)

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69 protocols using western lightning

Protein Extraction and Western Blot Analysis

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Samples were homogenized, centrifuged, assayed for protein content as described above, diluted in Laemmli sample buffer containing dithiothreitol, and boiled for 5 minutes. Samples were then analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis as described previously [20 (link)]. Samples were run on 4–20% Tris-HEPES gels (Pierce) and then transferred onto nitrocellulose membranes. After transfer, membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween. Proteins on the nitrocellulose membranes were probed with primary and secondary antibodies described in the ‘Materials’ section and then visualized using enhanced chemiluminescence (Western Lightning; PerkinElmer, Waltham, MA, USA). Western blots were quantified using TotalLab software purchased from TotalLab Nonlinear Dynamics (Newcastle on Tyre, UK). For probing ATM and P-ATM, samples were run on 3–8% Tris-Acetate gels (Invitrogen, Carlsbad, CA, USA) alongside HiMark (Invitrogen) molecular weight markers.

Spears L.D., Renth A.L., McKuin M.R., Kennedy A.R., Andrisse S., Briggs N.E, & Fisher J.S. (2017). A role for ataxia telangiectasia mutated in insulin-independent stimulation of glucose transport. Trends in cell & molecular biology, 12, 49-56.

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Western Blot Analysis of UGT1A1

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Whole tissue lysate (25 µg) was separated on 4%-12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA), and the protein transferred to nitrocellulose (Millipore). Following blocking with 5% nonfat dry milk in Tris-buffered saline solution (10 mM Tris [pH 8.0], 150 mM NaCl, 0.05%Tween20) for1 h, membranes were incubated with primary anti-UGT1A1 antibody overnight at 4 °C followed by three washes with Tris-buffered saline solution. Horseradish peroxidase-conjugated secondary antibody was added at room temperature for 1 h with gentle shaking. After application of chemiluminescence reagents (Western Lightning from Perkin Elmer Life Sciences), images were obtained in a Bio-Rad Universal Hood II equipped with a ChemiDox XRS imaging system.

Liu M., Chen S., Yueh M.F., Fujiwara R., Konopnicki C., Hao H, & Tukey R.H. (2016). Cadmium and arsenic override NF-κB developmental regulation of the intestinal UGT1A1 gene and control of hyperbilirubinemia. Biochemical pharmacology, 110-111, 37-46.

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Western Blot Analysis of Cell Lysates

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Whole cell lysates were prepared in radioimmunoprecipitation assay buffer (RIPA, Boston BioProducts), separated by SDS-PAGE at 100V using a Bio-Rad 4–20% TGX gel (Bio-Rad), blocked with 5% Milk-TBS-T for standard western blotting, 5% BSA for phospo-VEGFR2 blotting, and probed with antibodies overnight at 4°C. Peroxidase-conjugated secondary antibodies were incubated at RT for 1 hour, and bands were exposed with Western Lightning (PerkinElmer). Blots were imaged on a Fluorchem M (ProteinSimple). All antibodies and dilutions are presented in Supplementary Table 3. Uncropped western blots are presented in Supplementary Fig. 11.

Dunleavey J.M., Xiao L., Thompson J., Kim M.M., Shields J.M., Shelton S.E., Irvin D.M., Brings V.E., Ollila D., Brekken R.A., Dayton P.A., Melero-Martin J.M, & Dudley A.C. (2014). Vascular channels formed by subpopulations of PECAM1+ melanoma cells. Nature communications, 5, 5200.

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Quantitative Western Blotting of PTEN

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Whole cell extracts were prepared as previously described [22 (link)]. Protein (30 μg) was analyzed on a Novex^® 4–12% Tris-Bis gradient polyacrylamide gel and immunoblot analysis performed as described previously [32 (link)]. Primary antibodies used were β-actin (1:200) produced in mouse (C4:Santa Cruz Biotechnologies Inc.) and PTEN (1:1000) produced in rabbit (D4.3:Cell Signaling). After incubation in primary antibody overnight, the blot was washed as previously described [32 (link)] and incubated first in secondary anti-mouse IgG-HRP (1:1000) produced in goat (Santa Cruz Biotechnologies) and then with anti-rabbit IgG-HRP (1:1000) produced in goat (Cell Signaling) for 1 hour at room temperature. After washing bands were developed using the chemiluminescent reagents from Western Lightning^® (Perkin Elmer, Boston, MA). Western blots were exposed and bands quantitated using the ODYSSEY^® Fc Imaging System (LI-COR Biosciences, Lincoln, NE).

Budd W.T., Seashols-Williams S.J., Clark G.C., Weaver D., Calvert V., Petricoin E., Dragoescu E.A., O’Hanlon K, & Zehner Z.E. (2015). Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis. PLoS ONE, 10(11), e0142373.

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Quantitative Analysis of NR1 in Optic Nerve Lysates

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Protein lysates of optic nerves from individual mice were prepared using the Precellys Ceramic Kit 1.4 mm and the Precellys 24 homogenizer (Peqlab). Nerves were homogenized in 150 μl sucrose buffer (in mM: 320 sucrose, 10 Tris [pH 7.4], 1 NaHCO₃, and 1 MgCl₂) and protease inhibitors (Complete tablets, Roche). For Western blotting, 30 μg protein lysate was size-separated on 12% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes following instructions from BioRad. Primary antibodies to NR1 (1:500, Millipore Cat# MAB363, RRID: AB_94946) and GAPDH (1:2000, Enzo Life Sciences Cat# ADI-CSA-335-E, RRID: AB_2039148) were diluted in blocking buffer (5% milk) and incubated overnight at 4°C. Membranes were washed in 0.05% Tween prepared in phosphate buffer (PBS-T) followed by incubation with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected with an enhanced chemiluminescence kit (Western Lightning, PerkinElmer) according to the manufacturer’s instructions. Exposed ECL films (Amersham Biosciences) were scanned at grayscale (300 dpi resolution) using a regular image scanner, followed by densitometric analysis with ImageJ. The peak intensity for NR1 was normalized to the peak intensity of GAPDH.

Saab A.S., Tzvetavona I.D., Trevisiol A., Baltan S., Dibaj P., Kusch K., Möbius W., Goetze B., Jahn H.M., Huang W., Steffens H., Schomburg E.D., Pérez-Samartín A., Pérez-Cerdá F., Bakhtiari D., Matute C., Löwel S., Griesinger C., Hirrlinger J., Kirchhoff F, & Nave K.A. (2016). Oligodendroglial NMDA Receptors Regulate Glucose Import and Axonal Energy Metabolism. Neuron, 91(1), 119-132.

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Quantification of Cell-Associated Vitronectin

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Cell-associated vitronectin was quantified by Western blot. HUVEC, BBmVEC, PAEC, and PSMC samples were scraped off the surface in ice-cold lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 2 mM NaVO₄, 2 mM PMSF, 50 mM NaF, 10% glycerol, complete protease inhibitor (Roche), pH 7.4). Cell lysates were normalized for protein content, separated by SDS-PAGE on a 4–12% Bis-Tris gel (Life Technologies), and transferred to nitrocellulose membranes using the Invitrogen iBlot system. Membranes were incubated overnight at 4°C with vitronectin primary antibody (sc-15332, Santa Cruz), followed by an anti-rabbit secondary horseradish peroxidase-conjugated antibody (Promega) for 2 hours at room temperature. Protein bands were detected using an enhanced chemiluminescence kit (Western Lightning, PerkinElmer) and visualized with a Fluorchem digital imager (Alpha Innotech). Both native and multimeric vitronectin exhibited a 65/75 kDa doublet under reducing conditions, as previously reported [37 (link)]. Doublet intensity was quantified using AlphaEase FC software.

Mathew J.G., Basehore S, & Clyne A.M. (2017). Fluid Shear Stress and Fibroblast Growth Factor-2 Increase Endothelial Cell-Associated Vitronectin. Applied Bionics and Biomechanics, 2017, 9040161.

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Apolipoprotein A-I Remodeling Assay

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Remodeling of HDL in plasma was assessed after addition of peptides (final concentration 0.5 mg/mL) to pooled human plasma and incubated at 37°C for 1 h. Samples (10 ul) were mixed with 10 ul of 2X Native Tris-Glycine Sample Buffer (Novex/Thermo-Fisher, Carlsbad, CA) and electrophoresed on a native Tris-Glycine 10-20% acrylamide Novex WedgeWell gel and blotted onto PVDF as described earlier [25 ]. After blocking, the membrane was incubated with anti-human ApoA-I antibody preconjugated to HRP (Meridian Life Sciences, Memphis, TN), washed, and detected with Western Lightning (Perkin-Elmer, Waltham, MA).

Islam R., Sviridov D.O., Drake S.K., Tunyi J., Abdoulaeva G., Freeman L.A., Pastor R.W, & Remaley A.T. (2020). Incorporation of α-Methylated Amino Acids into Apolipoprotein A-I Mimetic Peptides Improves their Helicity and Cholesterol Efflux Potential. Biochemical and biophysical research communications, 526(2), 349-354.

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Comprehensive Western Blot Analysis

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For Western blot assay, cells and tissues were lysed in standard RIPA buffer. Antibodies for p-AKT, p-S6K, p-S6, P-4EBP1, 4EBP1, cyclin A, Snail, and CD31 were purchased from Cell Signaling. Antibody for actin was from Sigma. Antibodies were detected using the enhanced chemiluminescence method (Western Lightning, PerkinElmer). Immunoblot signals were acquired with the LAS-3000 Imaging system (FujiFilm) and were analyzed with ImageJ software.

Dong Y., Gong Y., Kuo F., Makarov V., Reznik E., Nanjangud G.J., Aras O., Zhao H., Qu R., Fagin J.A., Sherman E.J., Xu B., Ghossein R., Chan T.A, & Ganly I. (2021). Targeting the mTOR Pathway in Hurthle Cell Carcinoma Results in Potent Anti-Tumor Activity. Molecular cancer therapeutics, 21(2), 382-394.

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Immunoblotting for Protein Quantification

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Protein quantification was carried out by immunoblotting using 1:1000 dilutions of antibodies for Acot7 (Abcam85151), ACSL1 (Cell Signalling 4047), Mitochondrial complex (Mitosciences601), VDAC (Cell SignalingS4866), UCP3 (Abcam4866) as described^{22 (link)}. Briefly, lysates were prepared in Laemmli, resolved by SDS-PAGE electrophoresis on either 7.5% or 10% gels and proteins transferred to PVDF membrane (Hybond-P). Membranes were blocked in 2% BSA-Tris buffered saline with 0.1% Tween and incubated with primary antibody. After incubation with the appropriate secondary anitbody bands were visualised by chemiluminescence (Western lightning, Perkin Elmer) with exposure to film (Super RX Fuji Photo Film).

Bakshi I., Brown S.H., Brandon A.E., Suryana E., Mitchell T.W., Turner N, & Cooney G.J. (2018). Increasing Acyl CoA thioesterase activity alters phospholipid profile without effect on insulin action in skeletal muscle of rats. Scientific Reports, 8, 13967.

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Western Blot Analysis of Signaling Pathways

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Cells were lysed in lysis buffer containing 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA, 5000 U/mL aprotinin, 20 mg/mL leupeptin, 1 mmol/L phenylmethylsulfonylfluoride, 2 mmol/L sodium orthovanadate, 50 mmol/L NaF, 5% glycerol, 10 mmol/L Tris-HCl (pH 7.4), and 2% SDS. Next, protein concentrations were measured using a protein assay kit (Catalog #: 5000001, Bio-Rad, Richmond, CA), and then equal amounts of protein were separated using SDS-PAGE and electroblotted onto nitrocellulose membranes, which were blocked (overnight at 4 °C) with 5% nonfat milk in 0.1% Tween-20/TBST. The membranes were immunoblotted with primary antibodies from Cell Signaling Technology (CST) against EGFR (#2085), p-EGFR (#11862), JAK2 (Catalog #: 3230), p-JAK2 (Catalog #: 3771), STAT3 (Catalog #: 9139), and p-STAT3 (Catalog #: 9145), washed with Tween-20/PBS, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After washing with Tween-20/PBS, protein bands on the membranes were visualized using an enhanced chemiluminescence detection system (Western Lightning, Perkin-Elmer, Norwalk, CT, USA).

Wang Q., Liu J., You Z., Yin Y., Liu L., Kang Y., Li S., Ning S., Li H., Gong Y., Xu S, & Pang D. (2021). LncRNA TINCR favors tumorigenesis via STAT3–TINCR–EGFR-feedback loop by recruiting DNMT1 and acting as a competing endogenous RNA in human breast cancer. Cell Death & Disease, 12(1), 83.

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