Supersignal west femto ecl kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SuperSignal West Femto ECL kit is a chemiluminescent substrate used for the detection of horseradish peroxidase (HRP)-labeled proteins in Western blotting applications. The kit provides a sensitive and quantitative method for detecting low-abundance proteins.

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Lab products found in correlation

Nitrocellulose membrane, by Bio-Rad (4 mentions) Ripa buffer, by Thermo Fisher Scientific (4 mentions) Trans blot turbo transfer system, by Bio-Rad (2 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Polyvinylidene fluoride (pvdf), by Cytiva (2 mentions) Superblock t20, by Abcam (1 mentions) Superblock t20, by Thermo Fisher Scientific (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti β actin, by Thermo Fisher Scientific (1 mentions) Bca assay kit, by Thermo Fisher Scientific (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions) Image lab, by Bio-Rad (1 mentions) Quick start bradford assay, by Bio-Rad (1 mentions) Tnf α, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Pparα, by Abcam (1 mentions) Ge amersham imager 600, by GE Healthcare (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

Spelling variants of this product

ECL kit (865 citations) SuperSignal West Femto (427 citations) SuperSignal ECL kit (18 citations) Femto ECL (17 citations) SuperSignal ECL (17 citations) SuperSignal West Femto ECL (10 citations)

12 protocols using supersignal west femto ecl kit

Western Blot Analysis of XFS-related Proteins

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Total protein was extracted from iris and ciliary body tissues of 6 eyes with XFS and 6 normal eyes using RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS). Protein concentrations were determined by the Micro-BCA protein assay kit (Thermo Scientific). Proteins (10 μg per lane) were separated by 4–15% SDS-polyacrylamide gel electrophoresis under reducing conditions (6% DTT) and transferred onto nitrocellulose membranes with the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with SuperBlock T20 (Thermo Scientific) for 30 minutes and incubated for 1h at room temperature or overnight at 4°C with antibodies against POMP (Abcam) and TMEM136 (Abcam) diluted in PBST/10% SuperBlock T20. Equal loading was verified with mouse anti-human p- actin antibody (clone AC-15; Sigma-Aldrich) in PBST/10% SuperBlock T20. In negative control experiments, the primary antibody was replaced by PBST. Immunodetection was performed with a horseradish peroxidase-conjugated secondary antibody in PBST/10% SuperBlock T20 and the Super Signal West Femto ECL kit (Thermo Scientific), and band intensity was analysed by computerized densitometry.
Immunohistochemistry of human eye tissues follow routine laboratory procedures, and are appended in Supplementary Note.

Aung T., Ozaki M., Lee M.C., Schlötzer-Schrehardt U., Thorleifsson G., Mizoguchi T., Igo RP J.r., Haripriya A., Williams S.E., Astakhov Y.S., Orr A.C., Burdon K.P., Nakano S., Mori K., Abu-Amero K., Hauser M., Li Z., Prakadeeswari G., Bailey J.N., Cherecheanu A.P., Kang J.H., Nelson S., Hayashi K., Manabe S.I., Kazama S., Zarnowski T., Inoue K., Irkec M., Coca-Prados M., Sugiyama K., Järvelä I., Schlottmann P., Lerner S.F., Lamari H., Nilgün Y., Bikbov M., Park K.H., Cha S.C., Yamashiro K., Zenteno J.C., Jonas J.B., Kumar R.S., Perera S.A., Chan A.S., Kobakhidze N., George R., Vijaya L., Do T., Edward D.P., Marcos L.D., Pakravan M., Moghimi S., Ideta R., Bach-Holm D., Kappelgaard P., Wirostko B., Thomas S., Gaston D., Bedard K., Greer W.L., Yang Z., Chen X., Huang L., Sang J., Jia H., Jia L., Qiao C., Zhang H., Liu X., Zhao B., Wang Y.X., Xu L., Leruez S., Reynier P., Chichua G., Tabagari S., Uebe S., Zenkel M., Berner D., Mossböck G., Weisschuh N., Hoja U., Welge-Luessen U.C., Mardin C., Founti P., Chatzikyriakidou A., Pappas T., Anastasopoulos E., Lambropoulos A., Ghosh A., Shetty R., Porporato N., Saravanan V., Venkatesh R., Shivkumar C., Kalpana N., Sarangapani S., Kanavi M.R., Beni A.N., Yazdani S., lashay A., Naderifar H., Khatibi N., Fea A., Lavia C., Dallorto L., Rolle T., Frezzotti P., Paoli D., Salvi E., Manunta P., Mori Y., Miyata K., Higashide T., Chihara E., Ishiko S., Yoshida A., Yanagi M., Kiuchi Y., Ohashi T., Sakurai T., Sugimoto T., Chuman H., Aihara M., Inatani M., Miyake M., Gotoh N., Matsuda F., Yoshimura N., Ikeda Y., Ueno M., Sotozono C., Jeoung J.W., Sagong M., Park K.H., Ahn J., Cruz-Aguilar M., Ezzouhairi S.M., Rafei A., Chong Y.F., Ng X.Y., Goh S.R., Chen Y., Yong V.H., Khan M.I., Olawoye O.O., Ashaye A.O., Ugbede I., Onakoya A., Kizor-Akaraiwe N., Teekhasaenee C., Suwan Y., Supakontanasan W., Okeke S., Uche N., Asimadu I., Ayub H., Akhtar F., Kosior-Jarecka E., Lukasik U., Lischinsky I., Castro V., Grossmann R.P., Megevand G.S., Roy S., Dervan E., Silke E., Rao A., Sahay P., Fornero P., Cuello O., Sivori D., Zompa T., Mills R.A., Souzeau E., Mitchell P., Wang J.J., Hewitt A.W., Coote M., Crowston J.G., Astakhov S.Y., Akopov E.L., Emelyanov A., Vysochinskaya V., Kazakbaeva G., Fayzrakhmanov R., Al-Obeidan S.A., Owaidhah O., Aljasim L.A., Chowbay B., Foo J.N., Soh R.Q., Sim K.S., Xie Z., Cheong A.W., Mok S.Q., Soo H.M., Chen X.Y., Peh S.Q., Heng K.K., Husain R., Ho S.L., Hillmer A.M., Cheng C.Y., Escudero-Domínguez F.A., González-Sarmiento R., Martinon-Torres F., Salas A., Pathanapitoon K., Hansapinyo L., Wanichwecharugruang B., Kitnarong N., Sakuntabhai A., Nguyễn H.X., Nguyễn G.T., Nguyễn T.V., Zenz W., Binder A., Klobassa D.S., Hibberd M.L., Davila S., Herms S., Nöthen M.M., Moebus S., Rautenbach R.M., Ziskind A., Carmichael T.R., Ramsay M., Álvarez L., García M., González-Iglesias H., Rodríguez-Calvo P.P., Cueto L.F., Oguz Ç., Tamcelik N., Atalay E., Batu B., Aktas D., Kasim B., Wilson M.R., Coleman A.L., Liu Y., Challa P., Herndon L., Kuchtey R.W., Kuchtey J., Curtin K., Chaya C.J., Crandall A., Zangwill L.M., Wong T.Y., Nakano M., Kinoshita S., den Hollander A.I., Vesti E., Fingert J.H., Lee R.K., Sit A.J., Shingleton B.J., Wang N., Cusi D., Qamar R., Kraft P., Pericak-Vance M.A., Raychaudhuri S., Heegaard S., Kivelä T., Reis A., Kruse F.E., Weinreb R.N., Pasquale L.R., Haines J.L., Thorsteinsdottir U., Jonasson F., Allingham R.R., Milea D., Ritch R., Kubota T., Tashiro K., Vithana E.N., Micheal S., Topouzis F., Craig J.E., Dubina M., Sundaresan P., Stefansson K., Wiggs J.L., Pasutto F, & Khor C.C. (2017). Genetic association study of exfoliation syndrome identifies a protective rare variant at LOXL1 and five new susceptibility loci. Nature genetics, 49(7), 993-1004.

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CFTR Protein Expression Analysis in Rat Tissues

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Intestinal tissue was homogenized in PBS on ice followed by lysis in RIPA buffer (Thermo Fisher Scientific, Rockford, IL, United States) with Halt protease inhibitor cocktail (Thermo Fisher Scientific), as previously described (Tuggle et al., 2014 (link)). Protein quantification was performed using the BCA assay kit (Thermo Fisher Scientific). Samples were mixed with 4× sample buffer and incubated at 37°C for 10 min. Equal amounts of protein (20 μg) were loaded for electrophoresis. Wild type Sprague-Dawley rat trachea and lung extract were used as positive controls for CFTR detection. Membranes were then blocked in 5% non-fat dry milk dissolved in PBST, followed by incubation with monoclonal anti-CFTR (1:3,000; UNC 596) and mouse monoclonal anti-β-actin (1:5,000; Thermo Fisher Scientific), and subsequently goat anti-mouse secondary antibody conjugated to HRP (1:10,000; Dako North America, Inc.). Images were captured by ChemiDocXRS (Bio-Rad) using SuperSignal West Femto ECL kit (Thermo Fisher Scientific).

Sharma J., Abbott J., Klaskala L., Zhao G., Birket S.E, & Rowe S.M. (2020). A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay. Frontiers in Physiology, 11, 611294.

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Western Blotting Methodology for Protein Analysis

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Western blotting was performed similar to [112 (link),113 (link)]. Briefly, cells and tissues were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1 mM Na₃VO₄, 1 mM NaF, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture, pH 8.0) and boiled in 50 mM Tris, 2% (w/v) SDS, 3.34% (v/v) glycerol, and 16.67 mM β-mercaptoethanol. Proteins were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Supported, Hercules, CA, USA). Antibodies used in this study were diluted in 5% BSA solution and are shown in Table 2. The secondary antibody was an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA). Labeled proteins were detected using the Chemidoc Touch Imaging System with Supersignal West Pico and Supersignal West Femto ECL Kit (Thermo Fisher Scientific, Waltham, MA, USA). Blots were quantified using densitometry using Image Lab (Bio-Rad) software (version 6.1). All blots are shown in the Supplementary Materials.

Ujlaki G., Kovács T., Vida A., Kókai E., Rauch B., Schwarcz S., Mikó E., Janka E., Sipos A., Hegedűs C., Uray K., Nagy P, & Bai P. (2023). Identification of Bacterial Metabolites Modulating Breast Cancer Cell Proliferation and Epithelial-Mesenchymal Transition. Molecules, 28(15), 5898.

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Western Blot Analysis of Protein Expression

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Target protein expression was determined by Western blotting as described elsewhere. Briefly, HepG2 cells were lysed in RIPA buffer, and extracted proteins were quantified using Quick Start Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were resolved on 12% polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked and treated with primary Abs against TNF-α (Cell Signaling Technology; Cat#: 6945, Danvers, MA, USA), PPARα (Abcam; Cat#: ab3484, Cambridge, UK), or β-actin (Cell Signaling Technology; Cat#: 3700T, Danvers, MA, USA), followed by treatment with HRP-linked secondary Ab (Promega, Madison, WI, USA). Protein bands were developed using the SuperSignal West Femto ECL kit (Thermo Scientific, Waltham, MA, USA), and images were captured (ChemiDoc MP imaging system, Bio-Rad, Hercules, CA, USA).

Al-Rashed F., Arefanian H., Madhoun A.A., Bahman F., Sindhu S., AlSaeed H., Jacob T., Thomas R., Al-Roub A., Alzaid F., Malik M.Z., Nizam R., Thanaraj T.A., Al-Mulla F., Hannun Y.A, & Ahmad R. (2024). Neutral Sphingomyelinase 2 Inhibition Limits Hepatic Steatosis and Inflammation. Cells, 13(5), 463.

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Western Blot Analysis of Protein Samples

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An equal amount of protein (50–100 μg) samples was run on SDS–PAGE and transferred to PVDF membrane (Millipore). The membranes were then blocked in 5% skimmed milk in 0.1% TBS-Tween 20 at RT for 1 h on the rocker. Blots were cut according to molecular weights indicated by a pre-stained ladder (Abcam), and appropriate primary antibodies were added and incubated overnight (12–16 h) at 4°C. Blots were washed and incubated with appropriate secondary antibodies. After the secondary antibody incubation, the blots were washed thrice, and the Supersignal West Pico PLUS or Supersignal West Femto ECL kit (Thermo Fisher Scientific) was used to detect the bands on GE Amersham Imager 600. All antibodies, chemicals, and reagents used in this study are given in Table S2.

Table S2 Reagents/resources used in the study.

Usha Satheesan S., Chowdhury S, & Kolthur-Seetharam U. (2024). Metabolic and circadian inputs encode anticipatory biogenesis of hepatic fed microRNAs. Life Science Alliance, 7(5), e202302180.

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Western Blot Analysis of CFTR in Rat Lung

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Lung tissue was homogenized in TBS on ice followed by lysis in RIPA buffer (ThermoScientific, Rockford, Il) with Halt protease inhibitor cocktail (ThermoScientific). Protein was quantitated using the BCA assay (ThermoScientific), samples mixed with 4× sample buffer, and incubated at 37°C for 10 minutes. Equal amounts of protein (20 µg) were loaded into each lane, resolved by SDS-PAGE, and blotted onto PVDF membranes. Wild-type Sprague-Dawley rat lung extract (Sc-2396, Santa Cruz Biotechnology, Inc, Dallas, Tx) was used as an additional positive control for CFTR detection. Blocking was with 1% rabbit serum followed by incubation with goat anti-CFTR primary antibody (1∶200 Sc-8909) overnight at 4°C, and subsequent rabbit anti-goat HRP conjugated secondary antibody (1∶5000 Sc-2768) for 1 hour at room temperature. Labeled proteins were detected using SuperSignal West Femto ECL kit (ThermoScientific).

Tuggle K.L., Birket S.E., Cui X., Hong J., Warren J., Reid L., Chambers A., Ji D., Gamber K., Chu K.K., Tearney G., Tang L.P., Fortenberry J.A., Du M., Cadillac J.M., Bedwell D.M., Rowe S.M., Sorscher E.J, & Fanucchi M.V. (2014). Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats. PLoS ONE, 9(3), e91253.

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Western Blot Analysis of Insulin Signaling

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Protein lysates were prepared from isolated tissue using RIPA lysis buffer with added Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Equal amounts of protein lysate were loaded in TGX Precast gels (Bio-Rad) or in-house-made 7% polyacrylamide gels, then subjected to SDS–PAGE. Protein was transferred onto PVDF membranes using the Trans Blot Turbo transfer system (Bio-Rad). After transfer, blots were blocked for 1–3 h in 5% dry milk in TBST at room temperature, then probed using anti-phospho insulin receptor β (Tyr1150/1151) (19H7) (1:1,000 dilution, Cell Signaling, 3024), anti-insulin receptor β (1:5,000 dilution, Novus Biologicals, NBP2 12793), anti-Glut4 (1:1,000 dilution, Alomone Labs AGT, 024), anti-β actin HRP (1:15,000 dilution, Sigma, A3854) and Amersham ECL anti-rabbit IgG HRP (1:5,000 dilution, GE Healthcare, NA934V). In some experiments, primary and secondary antibodies were diluted in SignalBoost Immunoreaction Enhancer kit solutions (Millipore Sigma). Blots were exposed using Western Lightning Plus ECL kit (PerkinElmer) or SuperSignal West Femto ECL kit (Thermo Scientific). Images were collected on the ChemiDoc Touch imagining system and analyzed using ImageLab software (Bio-Rad).

Iker Etchegaray J., Kelley S., Penberthy K., Karvelyte L., Nagasaka Y., Gasperino S., Paul S., Seshadri V., Raymond M., Marco A.R., Pinney J., Stremska M., Barron B., Lucas C., Wase N., Fan Y., Unanue E., Kundu B., Burstyn-Cohen T., Perry J., Ambati J, & Ravichandran K.S. (2023). Phagocytosis in the retina promotes local insulin production in the eye. Nature metabolism, 5(2), 207-218.

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Protein Quantification and Western Blot Analysis

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Protein concentrations were determined by Bradford Protein Assay. Transiently transfected cells and human skin fibroblasts were rinsed twice in ice-cold PBS prior to lysis in lysis buffer (5M NaCl, NP-40, 1M Tris; pH 8.0), 0.5M EDTA, H2O) supplemented with 1 mM Na3VO4, 1 mM PMSF, 10 mM DTT and protease inhibitors (Complete; Roche, Basle, Switzerland). Protein lysates were boiled at 95°C for 5 minutes prior to gel loading. In all experiments, SDS-PAGE (6 and 8%) was followed by protein transfer onto PVDF (Amersham) or nitrocellulose membranes (BioRad Laboratories) for immunoblot analysis. Mouse monoclonal anti-β actin, anti-α tubulin or anti GAPDH were used as internal controls. Proteins were detected with either ECL detection system or SuperSignal West Femto ECL kit (Thermo Scientific). Densitometry analysis of western blot bands was performed using Image Lab 5.0 software (Bio-Rad Laboratories).

Oldoni F., van Capelleveen J.C., Dalila N., Wolters J.C., Heeren J., Sinke R.J., Hui D.Y., Dallinga-Thie G.M., Frikke-Schmidt R., Hovingh K.G., van de Sluis B., Tybjærg-Hansen A, & Kuivenhoven J.A. (2018). Naturally Occurring Variants in LRP1 (Low-Density Lipoprotein Receptor–Related Protein 1) Affect HDL (High-Density Lipoprotein) Metabolism Through ABCA1 (ATP-Binding Cassette A1) and SR-B1 (Scavenger Receptor Class B Type 1) in Humans. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(7), 1440-1453.

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Western Blot Analysis of Transfected Cells

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We cultured hTERT‐RPE1 cells in DMEM/F12 Glutamax medium with 10% foetal bovine serum (Life Technologies). We grew the cells in six well‐plates to reach an 80% confluence, transfected them with 2.5 µg expression plasmids using lipofectamine 3000 (Invitrogen L3000‐008), collected and lysed them 48 h after transfection using the M‐PER mammalian protein extraction reagent (Thermo 78503). We added MG132 (final concentration 10 µM, 0.1% DMSO) 16 h prior to cell lysis. We loaded equal amounts of protein extracts onto NuPage MOPS 4–12% polyacrylamide gels, transferred them onto nitrocellulose membranes and probed the upper halves of the membranes with an anti‐tubulin monoclonal antibody (1/5000, Sigma B512). We probed the lower halves with either an anti‐Myc monoclonal antibody (1/1000, Cell signalling 9B11) or an anti‐Flag antibody (1/700, Sigma 7425). We revealed the Flag blots with the same antibody as described above and a SuperSignal West Femto ECL kit (Thermo 34095). We revealed the Myc and the tubulin blots with an HRP‐coupled goat anti‐mouse antibody (1/1500, DAKO P0447) and the SuperSignal or the regular ECL detection kit, respectively.

Robert T., Dock‐Bregeon A, & Colas P. (2021). Functional characterization of CDK10 and cyclin M truncated variants causing severe developmental disorders. Molecular Genetics & Genomic Medicine, 9(10), e1782.

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Protein Quantification and Western Blot Analysis

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Protein concentrations were determined by Bradford Protein Assay. Transiently transfected cells and human skin fibroblasts were rinsed twice in ice-cold PBS before lysis in lysis buffer (5 mol/L NaCl, NP-40, 1 mol/L Tris; pH 8.0, 0.5 mol/L EDTA, H₂O) supplemented with 1 mmol/L Na₃VO₄, 1 mmol/L PMSF (phenylmethylsulfonyl fluoride), 10 mmol/L dithiothreitol, and protease inhibitors (Complete; Roche, Basle, Switzerland). Protein lysates were boiled at 95°C for 5 minutes before gel loading. In all experiments, SDS-PAGE (6% and 8%) was followed by protein transfer onto polyvinylidene fluoride (Amersham) or nitrocellulose membranes (Bio-Rad Laboratories) for immunoblot analysis. Mouse monoclonal anti–β-actin, anti–α-tubulin, or anti-GAPDH was used as internal controls. Proteins were detected with either ECL detection system or SuperSignal West Femto ECL kit (Thermo Scientific). Densitometry analysis of Western blot bands was performed using Image Laboratory 5.0 software (Bio-Rad Laboratories).

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