Nanoluc luciferase

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NanoLuc luciferase is a small luciferase enzyme derived from the deep sea shrimp Oplophorus gracilirostris. It catalyzes the oxidation of its substrate to produce luminescence, which can be used as a reporter for gene expression and other biological processes.

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Lab products found in correlation

Nano glo luciferase assay reagent, by Promega (4 mentions) Ppk cmv f4 fusion vector, by PromoCell (4 mentions) Victor x multilabel plate reader, by PerkinElmer (3 mentions) 96 well microfilter plate, by Merck Group (3 mentions) Nano glo luciferase assay system, by Promega (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Nanoglo reagent, by Promega (1 mentions) Anti flag hrp, by Merck Group (1 mentions) Nano glo luciferase assay substrate, by Promega (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Lumat lb 9507, by Berthold Technologies (1 mentions) Jetprime reagent, by Polyplus Transfection (1 mentions) Cell culture lysis reagent, by Promega (1 mentions) Nano glow luciferase assay system, by Promega (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Pcdna3.1 plasmid, by Thermo Fisher Scientific (1 mentions) Pnl1.1 plasmid, by Promega (1 mentions) Site directed mutagenesis, by Agilent Technologies (1 mentions) Gibson assembly, by New England Biolabs (1 mentions) Prism 8, by GraphPad (1 mentions)

Close spelling variants of this product

NanoLuc (35 citations) NanoLuc Luciferase Assay (11 citations) NanoLuc luciferase assay kit (4 citations) NanoLuc luciferase reporter plasmid (3 citations) NanoLuc luciferase reporter gene (2 citations) NanoLuc luciferase gene (2 citations)

19 protocols using nanoluc luciferase

Quantifying Protein-Protein Interactions via LUMIER

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LUminescence-based Mammalian IntERactome (LUMIER) assay was carried out by transfecting HEK293 cells in 96-well plates with plasmids that expressed each client protein fused to NanoLuc luciferase (Promega) along with 3XFLAG-tagged HSP90α and HSP90β. After 18 hours, cells were washed with cold PBS and lysed with 100 μl TGNET complete buffer, then centrifuged at maximum speed for 15 minutes at 4°C. The resulting lysates were transferred to an anti-FLAG antibody-coated plate (Sigma) and incubated at 4°C for 2 hours with gentle shaking. The plates were washed with TGNET. Nano-Glo reagent (Promega) was added and luciferase activity was quantified using a plate reader (Perkin-Elmer). To normalize for 3XFLAG-HSP90 levels, the plates were washed again with TGNET and anti-FLAG-HRP (Sigma-Aldrich) was added. The plates were once again incubated at 4°C, washed and assayed for HRP luminescence activity. The experimental relative interaction strength for each client protein interaction was determined by dividing the NanoLuc readout by the HRP readout value. Each sample was assayed 3 times with 3 replicates. Standard deviations are represented by error bars. A two-tailed T-test was used to determine statistical significance. All calculations were performed in Excel (Microsoft).

Prince T.L., Kijima T., Tatokoro M., Lee S., Tsutsumi S., Yim K., Rivas C., Alarcon S., Schwartz H., Khamit-Kush K., Scroggins B.T., Beebe K., Trepel J.B, & Neckers L. (2015). Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants. PLoS ONE, 10(10), e0141786.

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Measuring Autoantibodies Against IFN-α Subtypes

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Levels of autoantibodies against IFN-α subtypes were measured in luciferase-based immunoprecipitation assay (LIPS), as previously described (9). IFNA1, IFNA2, IFNA8, and IFNA21 sequences were inserted into a modified pPK-CMV-F4 fusion vector (PromoCell GmbH, Germany), in which the firefly luciferase replaced the NanoLuc luciferase (Promega, USA). The resulting constructs were used to transfect HEK293 cells and the IFNA-luciferase fusion proteins were collected in the tissue culture supernatant. For autoantibody screening, we combined 2x10⁶ luminescence units (LU) of IFNA1, IFNA2, IFNA8 and IFNA21 in a single IP reaction mixture (pool 1), and IFNA4, IFNA5, IFNA6 and IFNA7 in another IP reaction mixture (pool 2). Serum samples were incubated with Protein G agarose beads (Exalpha Biologicals, USA) at room temperature for 1 hour in a 96-well microfilter plate (Merck Millipore, Germany), and we then added 2x10⁶ luminescence units (LU) of antigen and incubated for another hour. Each sample was tested once. The plate was washed with a vacuum system and Nano-Glo® Luciferase Assay Reagent (Promega, USA) was added. Luminescence intensity was measured with a VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, USA). The results are expressed in arbitrary units (AU), as a fold-difference relative to the mean of the negative control samples.

Bastard P., Gervais A., Le Voyer T., Rosain J., Philippot Q., Manry J., Michailidis E., Hoffmann H.H., Eto S., Garcia-Prat M., Bizien L., Parra-Martínez A., Yang R., Haljasmägi L., Migaud M., Särekannu K., Maslovskaja J., de Prost N., Tandjaoui-Lambiotte Y., Luyt C.E., Amador-Borrero B., Gaudet A., Poissy J., Morel P., Richard P., Cognasse F., Troya J., Trouillet-Assant S., Belot A., Saker K., Garçon P., Rivière J.G., Lagier J.C., Gentile S., Rosen L.B., Shaw E., Morio T., Tanaka J., Dalmau D., Tharaux P.L., Sene D., Stepanian A., Megarbane B., Triantafyllia V., Fekkar A., Heath J.R., Franco J.L., Anaya J.M., Solé-Violán J., Imberti L., Biondi A., Bonfanti P., Castagnoli R., Delmonte O.M., Zhang Y., Snow A.L., Holland S.M., Biggs C., Moncada-Vélez M., Arias A.A., Lorenzo L., Boucherit S., Coulibaly B., Anglicheau D., Planas A.M., Haerynck F., Duvlis S., Nussbaum R.L., Ozcelik T., Keles S., Bousfiha A.A., El Bakkouri J., Ramirez-Santana C., Paul S., Pan-Hammarström Q., Hammarström L., Dupont A., Kurolap A., Metz C.N., Aiuti A., Casari G., Lampasona V., Ciceri F., Barreiros L.A., Dominguez-Garrido E., Vidigal M., Zatz M., van de Beek D., Sahanic S., Tancevski I., Stepanovskyy Y., Boyarchuk O., Nukui Y., Tsumura M., Vidaur L., Tangye S.G., Burrel S., Duffy D., Quintana-Murci L., Klocperk A., Kann N.Y., Shcherbina A., Lau Y.L., Leung D., Coulongeat M., Marlet J., Koning R., Reyes L.F., Chauvineau-Grenier A., Venet F., Monneret G., Nussenzweig M.C., Arrestier R., Boudhabhay I., Baris-Feldman H., Hagin D., Wauters J., Meyts I., Dyer A.H., Kennelly S.P., Bourke N.M., Halwani R., Sharif-Askari N.S., Dorgham K., Sallette J., Sedkaoui S.M., AlKhater S., Rigo-Bonnin R., Morandeira F., Roussel L., Vinh D.C., Ostrowski S.R., Condino-Neto A., Prando C., Bonradenko A., Spaan A.N., Gilardin L., Fellay J., Lyonnet S., Bilguvar K., Lifton R.P., Mane S., Anderson M.S., Boisson B., Béziat V., Zhang S.Y., Vandreakos E., Hermine O., Pujol A., Peterson P., Mogensen T.H., Rowen L., Mond J., Debette S., de Lamballerie X., Duval X., Mentré F., Zins M., Soler-Palacin P., Colobran R., Gorochov G., Solanich X., Susen S., Martinez-Picado J., Raoult D., Vasse M., Gregersen P.K., Piemonti L., Rodríguez-Gallego C., Notarangelo L.D., Su H.C., Kisand K., Okada S., Puel A., Jouanguy E., Rice C.M., Tiberghien P., Zhang Q., Cobat A., Abel L., Casanova J.L., Bigio B., Boucherit S., de la Chapelle A., Chen J., Chrabieh M., Coulibaly B., Liu D., Nemirowskaya Y., Cruz I.M., Materna M., Pelet S., Seeleuthner Y., Thibault C., Liu Z., Abad J., Accordino G., Achille C., Aguilera-Albesa S., Aguiló-Cucurull A., Aiuti A., Özkan E.A., Darazam I.A., Roblero Albisures J.A., Aldave J.C., Ramos M.A., Khan T.A., Aliberti A., Nadji S.A., Alkan G., Alkhater S.A., Allardet-Servent J., Allende L.M., Alonso-Arias R., Alshahrani M.S., Alsina L., Alyanakian M.A., Borrero B.A., Amoura Z., Antolí A., Arrestier R., Aubart M., Auguet T., Avramenko I., Aytekin G., Azot A., Bahram S., Bajolle F., Baldanti F., Baldolli A., Ballester M., Feldman H.B., Barrou B., Barzagh F., Basso S., Bayhan G.I., Belot A., Bezrodnik L., Bilbao A., Blanchard-Rohner G., Blanco I., Blandinières A., Blázquez-Gamero D., Bleibtreu A., Bloomfield M., Bolivar-Prados M., Bondarenko A., Borghesi A., Borie R., Botdhlo-Nevers E., Bousfiha A.A., Bousquet A., Boutolleau D., Bouvattier C., Boyarchuk O., Bravais J., Briones M.L., Brunner M.E., Bruno R., Bueno M.R., Bukhari H., Bustamante J., Cáceres Agra J.J., Capra R., Carapito R., Carrabba M., Casari G., Casasnovas C., Caseris M., Cassaniti I., Castelle M., Castelli F., de Vera M.C., Castro M.V., Catherinot E., Celik J.B., Ceschi A., Chalumeau M., Charbit B., Cheng M.P., Clavé P., Clotet B., Codina A., Cohen Y., Colobran R., Comarmond C., Combes A., Comoli P., Corsico A.G., Coşkuner T., Cvetkovski A., Cyrus C., Dalmau D., Danion F., Darley D.R., Das V., Dauby N., Dauger S., De Munter P., de Pontual L., Dehban A., Delplancq G., Demoule A., Desguerre I., Di Sabatino A., Diehl J.L., Dobbelaere S., Domínguez-Garrido E., Dubost C., Ekwall O., Bozdemir Ş.E., Elnagdy M.H., Emiroglu M., Endo A., Erdeniz E.H., Aytekin S.E., Lasa M.P., Euvrard R., Fabio G., Faivre L., Falck A., Fartoukh M., Faure M., Arquero M.F., Ferrer R., Ferreres J., Flores C., Francois B., Fumadó V., Fung K.S., Fusco F., Gagro A., Solis B.G., Gaussem P., Gayretli Z., Gil-Herrera J., Gilardin L., Gatineau A.G., Girona-Alarcón M., Cifuentes Godínez K.A., Goffard J.C., Gonzales N., Gonzalez-Granado L.I., González-Montelongo R., Guerder A., Gülhan B., Gumucio V.D., Hanitsch L.G., Gunst J., Gut M., Hadjadj J., Haerynck F., Halwani R., Hammarström L., Hancerli S., Hariyan T., Hatipoglu N., Heppekcan D., Hernandez-Brito E., Ho P.K., Holanda-Peña M.S., Horcajada J.P., Hraiech S., Humbert L., Hung I.F., Iglesias A.D., Íñigo-Campos A., Jamme M., Arranz M.J., Jimeno M.T., Jordan I., Yüksek S.K., Kara Y.B., Karahan A., Karbuz A., Yasar K.K., Kasapcopur O., Kashimada K., Keles S., Demirkol Y.K., Kido Y., Kizil C., Kılıç A.O., Klocperk A., Koutsoukou A., Król Z.J., Ksouri H., Kuentz P., Kwan A.M., Kwan Y.W., Kwok J.S., Lagier J.C., Lam D.S., Lampropoulou V., Lanternier F., Lau Y.L., Le Bourgeois F., Leo Y.S., Lopez R.L., Leung D., Levin M., Levy M., Lévy R., Li Z., Lilleri D., Lima E.J., Linglart A., López-Collazo E., Lorenzo-Salazar J.M., Louapre C., Lubetzki C., Lung K.C., Luyt C.E., Lye D.C., Magnone C., Mansouri D., Marchioni E., Marioli C., Marjani M., Marques L., Pereira J.M., Martín-Nalda A., Pueyo D.M., Martinez-Picado J., Marzana I., Mata-Martínez C., Mathian A., Matos L.R., Matthews G.V., Mayaux J., McLaughlin-Garcia R., Meersseman P., Mège J.L., Mekontso-Dessap A., Melki I., Meloni F., Meritet J.F., Merlani P., Akcan Ö.M., Meyts I., Mezidi M., Migeotte I., Millereux M., Million M., Mirault T., Mircher C., Mirsaeidi M., Mizoguchi Y., Modi B.P., Mojoli F., Moncomble E., Melián A.M., Martinez A.M., Morandeira F., Morange P.E., Mordacq C., Morelle G., Mouly S.J., Muñoz-Barrera A., Nafati C., Nagashima S., Nakagama Y., Neven B., Neves J.F., Ng L.F., Ng Y.Y., Nielly H., Medina Y.N., Cuadros E.N., Ocejo-Vinyals J.G., Okamoto K., Oualha M., Ouedrani A., Özçelik T., Ozkaya-Parlakay A., Pagani M., Pan-Hammarström Q., Papadaki M., Parizot C., Parola P., Pascreau T., Paul S., Paz-Artal E., Pedraza S., González Pellecer N.C., Pellegrini S., de Diego R.P., Pérez-Fernández X.L., Philippe A., Philippot Q., Picod A., de Chambrun M.P., Piralla A., Planas-Serra L., Ploin D., Poissy J., Poncelet G., Poulakou G., Pouletty M.S., Pourshahnazari P., Qiu-Chen J.L., Quentric P., Rambaud T., Raoult D., Raoult V., Rebillat A.S., Redin C., Resmini L., Ricart P., Richard J.C., Rigo-Bonnin R., Rivet N., Rivière J.G., Rocamora-Blanch G., Rodero M.P., Rodrigo C., Rodriguez L.A., Rodriguez-Gallego C., Rodriguez-Palmero A., Romero C.S., Rothenbuhler A., Roux D., Rovina N., Rozenberg F., Ruch Y., Ruiz M., Ruiz del Prado M.Y., Ruiz-Rodriguez J.C., Sabater-Riera J., Saks K., Salagianni M., Sanchez O., Sánchez-Montalvá A., Sánchez-Ramón S., Schidlowski L., Schluter A., Schmidt J., Schmidt M., Schuetz C., Schweitzer C.E., Scolari F., Sediva A., Seijo L., Seminario A.G., Sene D., Seng P., Senoglu S., Seppänen M., Llovich A.S., Shahrooei M., Shcherbina A., Siguret V., Siouti E., Smadja D.M., Smith N., Sobh A., Solanich X., Solé-Violán J., Soler C., Soler-Palacín P., Sözeri B., Stella G.M., Stepanovskiy Y., Stoclin A., Taccone F., Tandjaoui-Lambiotte Y., Taupin J.L., Tavernier S.J., Tello L.V., Terrier B., Thiery G., Thorball C., THORN K., Thumerelle C., Tipu I., Tolstrup M., Tomasoni G., Toubiana J., Alvarez J.T., Triantafyllia V., Trouillet-Assant S., Troya J., Tsang O.T., Tserel L., Tso E.Y., Tucci A., Tüter Öz Ş.K., Ursini M.V., Utsumi T., Uzunhan Y., Vabres P., Valencia-Ramos J., Van Den Rym A.M., Vandernoot I., Velez-Santamaria V., Zuniga Veliz S.P., Vidigal M.C., Viel S., Vilain C., Vilaire-Meunier M.E., Villar-García J., Vincent A., Vogt G., Voiriot G., Volokha A., Vuotto F., Wauters E., Wauters J., Wu A.K., Wu T.C., Yahşi A., Yesilbas O., Yildiz M., Young B.E., Yükselmiş U., Zatz M., Zecca M., Zuccaro V., Jens V.P., Lambrecht B.N., Eva V.B., Cédric B., Levi H., Eric H., Bauters F., De Clercq J., Cathérine H., Hans S., Leslie N., Florkin B., Boulanger C., Vanderlinden D., Foti G., Bellani G., Citerio G., Contro E., Pesci A., Valsecchi M.G., Cazzaniga M., Danielson J.J., Dobbs K., Kashyap A., Ding L., Dalgard C.L., Sottini A., Quaresima V., Quiros-Roldan E., Rossi C., Bettini L.R., D’Angio’ M., Beretta I., Montagna D., Licari A., Marseglia G.L., Batten I., Reddy C., McElheron M., Noonan C., Connolly E., Fallon A., Storgaard M., Jørgensen S., Tolstrup M., Erikstrup C., Pedersen O.B., Sørensen E., Mikkelsen S., Dinh K.M., Larsen M.A., Paulsen I.W., Von Stemann J.H., Hansen M.B., Ostrowski S.R., Townsend L., Cheallaigh C.N., Bergin C., Martin-Loeches I., Dunne J., Conlon N., Bourke N., O'Farrelly C., Abel L., Allavena C., Andrejak C., Angoulvant F., Azoulay C., Bachelet D., Bartoli M., Basmaci R., Behilill S., Beluze M., Benech N., Benkerrou D., Bhavsar K., Bitker L., Bouadma L., Bouscambert-Duchamp M., Paz P.C., Cervantes-Gonzalez M., Chair A., Chirouze C., Coelho A., Cordel H., Couffignal C., Couffin-Cadiergues S., d’Ortenzio E., De Montmollin E., Debard A., Debray M.P., Deplanque D., Descamps D., Desvallée M., Diallo A., Diehl J.L., Diouf A., Dorival C., Dubos F., Duval X., Eloy P., Enouf V., Epaulard O., Esperou H., Esposito-Farese M., Etienne M., Garot D., Gault N., Gaymard A., Ghosn J., Gigante T., Gilg M., Goehringer F., Guedj J., Hoctin A., Hoffmann I., Houas I., Hulot J.S., Jaafoura S., Kafif O., Kaguelidou F., Kali S., Kerroumi Y., Khalil A., Khan C., Kimmoun A., Laine F., Laouénan C., Laribi S., Le M., Le Bris C., Le Gac S., Le Hingrat Q., Le Mestre S., Le Nagard H., Lemaignen A., Lemee V., Lescure F.X., Letrou S., Levy Y., Lina B., Lingas G., Lucet J.C., Machado M., Malvy D., Mambert M., Manuel A., Mentré F., Meziane A., Mouquet H., Mullaert J., Neant N., Nguyen D., Noret M., Papadopoulos A., Paul C., Peiffer-Smadja N., Peigne V., Petrov-Sanchez V., Peytavin G., Pham H., Picone O., Piquard V., Poissy J., Puéchal O., Rosa-Calatrava M., Rossignol B., Rossignol P., Roy C., Schneider M., Su R., Tardivon C., Tellier M.C., Téoulé F., Terrier O., Timsit J.F., Tual C., Tubiana S., Van Der Werf S., Vanel N., Veislinger A., Visseaux B., Wiedemann A., Yazdanpanah Y., Annereau J.P., Briseño-Roa L., Gribouval O., Pelet A., Abel L., Alcover A., Aschard H., Bousso P., Bourke N., Brodin P., Bruhns P., Cerf-Bensussan N., Cumano A., D’Enfert C., Deriano L., Dillies M.A., Di Santo J., Dromer F., Eberl G., Enninga J., Fellay J., Gomperts-Boneca I., Hasan M., Hedestam G.K., Hercberg S., Ingersoll M.A., Lantz O., Kenny R.A., Ménager M., Michel F., Mouquet H., O'Farrelly C., Patin E., Pellegrini S., Rausell A., Rieux-Laucat F., Rogge L., Fontes M., Sakuntabhai A., Schwartz O., Schwikowski B., Shorte S., Tangy F., Toubert A., Touvier M., Ungeheuer M.N., Zimmer C., Albert M.L., Duffy D., Quintana-Murci L., Alavoine L., Behillil S., Burdet C., Charpentier C., Dechanet A., Descamps D., Duval X., Ecobichon J.L., Enouf V., Frezouls W., Houhou N., Kafif O., Lehacaut J., Letrou S., Lina B., Lucet J.C., Manchon P., Nouroudine M., Piquard V., Quintin C., Thy M., Tubiana S., van der Werf S., Vignali V., Visseaux B., Yazdanpanah Y., Chahine A., Waucquier N., Migaud M.C., Deplanque D., Djossou F., Mergeay-Fabre M., Lucarelli A., Demar M., Bruneau L., Gérardin P., Maillot A., Payet C., Laviolle B., Laine F., Paris C., Desille-Dugast M., Fouchard J., Malvy D., Nguyen D., Pistone T., Perreau P., Gissot V., Le Goas C., Montagne S., Richard L., Chirouze C., Bouiller K., Desmarets M., Meunier A., Lefévre B., Jeulin H., Legrand K., Lomazzi S., Tardy B., Gagneux-Brunon A., Bertholon F., Botelho-Nevers E., Kouakam C., Leturque N., Roufai L., Amat K., Couffin-Cadiergues S., Espérou H., Hendou S., van 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(2021). Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths. Science Immunology, 6(62), eabl4340.

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Monitoring Bacterial Lysis via Fluorescence and Luciferase Assays

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To monitor bacterial lysis during the bacterial peptide treatments, we transformed E. coli BL21 with reporter plasmids expressing codon-optimized eGFP and NanoLuc^® Luciferase (Promega, Madison, WI, USA) genes under the control of the constitutive Pupp promoter. To monitor bacterial lysis using fluorescent microscopy, BL21 (pACYC184-GFP) cells were incubated with 10 µM of the antibacterial peptides, and microscopy was performed at time points 15, 30, and 60 min and at 4 h. All fluorescence images were taken at 488 nm excitation. The emission filter was 525/50 nM for cytoplasmic GFP. Phase-contrast images were also collected at the same time points. To monitor bacterial lysis in solutions by protein leakage, BL21 (pACYC184 nLuc) cells were washed with PBS (pH 7.4) three times, diluted with PBS to 10⁵ cfu/ml, and then incubated with peptides for 15, 30, and 60 min. After incubation, the intact bacterial cells were precipitated and the luciferase activity of the supernatant was examined using the Nano-Glo^® Luciferase Assay Substrate (Promega). The linearity of the assay was assessed using activity measurements of serial dilutions of the completely lysed BL21 (pACYC184 nLuc) bacterial culture. An undiluted bacterial culture was taken as 100%. Data were corrected to the background luciferase activity obtained with the supernatant of untreated cells.

Basu S., Sineva E., Nguyen L., Sikdar N., Park J.W., Sinev M., Kunta M, & Gupta G. (2022). Host-derived chimeric peptides clear the causative bacteria and augment host innate immunity during infection: A case study of HLB in citrus and fire blight in apple. Frontiers in Plant Science, 13, 929478.

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Nanoluc Luciferase Assay Protocol

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Cells were seeded at a density of 2 × 10⁵ cells per well in 24-wells dishes. Transfections were performed using jetPRIME^® reagent (Polyplus-transfection Inc., Illkirch, France) or Lipofectamine^® 3000 (Life Technologies, Carlsbad, CA, USA). Twenty-four to 48 hours after transfection, cells were lyzed in 1× Cell Culture Lysis Reagent (Promega). The activity of the Nanoluc^® Luciferase (Promega) gene reporter was measured using the Nano Glow^® Luciferase Assay System (Promega) according to manufacturer’s instructions and using a Lumat LB 9507 (Berthold Technologies, Pforzheim, Germany) luminometer.

Rothenberger S., Torriani G., Johansson M.U., Kunz S, & Engler O. (2016). Conserved Endonuclease Function of Hantavirus L Polymerase. Viruses, 8(5), 108.

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Tau Protein Interaction Screening in HuH-7 Cells

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Human liver cell line (HuH-7) was maintained in Dulbecco’s modified Eagle’s minimum essential medium (DMEM/F-12) containing 9% foetal bovine serum. Transfection of the plasmids pFN33_tau and pFC36_tau (human 441aa 2N4R tau isoform fused to split luciferase fragments LgBit and SmBit, respectively) or pNL1.1.TK[Nluc/TK] vector (full length constitutively-active NanoLuc luciferase, Promega, Mannheim, Germany) in HuH-7 cells was performed in 6-well plates using the Lipofectamine^® 2000 Transfection reagent (ThermoFisher Scientific, Schwerte, Germany) according to manufacturer’s recommendation. Briefly, 2.5 µg DNA in 250 µL Opti-MEM^TM have been mixed with 6.2 µL of Lipofectamine^® 2000 in 250 µL Opti-MEM^TM incubated for 20 min and added to the HuH-7 cells per well. After 24 h incubation period, Huh-7 cells were plated into 384-well plates (3000 cells per well, µclear/white #781093 greiner bio-one) for inhibitor treatment and luciferase assay.

Holzer M., Schade N., Opitz A., Hilbrich I., Stieler J., Vogel T., Neukel V., Oberstadt M., Totzke F., Schächtele C., Sippl W, & Hilgeroth A. (2018). Novel Protein Kinase Inhibitors Related to Tau Pathology Modulate Tau Protein-Self Interaction Using a Luciferase Complementation Assay. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 23(9), 2335.

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LIPS Assay for APECED Autoantibody Detection

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Luciferase based immunoprecipitation system assay was conducted on saliva samples (10 APECED patients and 10 controls) and on plasma samples (13 APECED patients and 7 controls). LIPS assay was performed as previously described (32 (link)). Briefly, IFN-α, IL-17A, IL-17F, and IL-22 coding sequences were cloned into modified pPK-CMV-F4 fusion vector (PromoCell GmbH, Germany) where Firefly luciferase was substituted with NanoLuc luciferase (Promega, United States). Cloned constructs were transfected into HEK293 cells and secreted luciferase fusion proteins were collected with culture medium after 48 h. IgG from plasma and saliva samples was captured onto Protein G Agarose beads (Exalpha Biologicals, United States) at room temperature for 1 h in 96-well microfilter plate (Merck Millipore, Germany). Next, 1 × 10⁶ luminescence units (LU) of antigen was added per well. After 1 h of incubation the unbound antigen was washed away with vacuum system and Nano-Glo^® Luciferase Assay Reagent (Promega, United States) was added. Luminescence intensity was measured by VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, United States). The results were expressed as luminescence units (LU) representing the fold over the mean of the heathy control samples.

Kaleviste E., Rühlemann M., Kärner J., Haljasmägi L., Tserel L., Org E., Trebušak Podkrajšek K., Battelino T., Bang C., Franke A., Peterson P, & Kisand K. (2020). IL-22 Paucity in APECED Is Associated With Mucosal and Microbial Alterations in Oral Cavity. Frontiers in Immunology, 11, 838.

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Constructing Luciferase-MS2CP Expression Vector

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To generate the luciferase-MS2CP expression vector, NanoLuc luciferase was amplified from the pNL1.1 plasmid (Promega) and cloned into the pCi-MS2 vector^{47 (link)} using PstI and BamHI restriction sites. A puromycin resistance gene was added to the plasmid using PvtI restriction site. To remove the FLAG tag present in the original plasmid, a stop codon was introduced between the MS2CP and the FLAG tag by site-directed mutagenesis (Agilent).
To generate customized RNA-10xMS2 constructs, the pCDNA3.1 plasmid (Thermo Fisher) was modified. First, additional restriction sites (BstBI, AgeI, ClaI, AscI, PacI, BglII, and SrfI) were incorporated between the KpnI and BamHI sites. Second, MS2 stem loops were inserted between the BamHI and BglII sites. Xist(A), (F), and (C) fragments were PCR-amplified from BAC 399K20 (covering chrX:100,578,985–100,773,006, mm9 genome assembly), and cloned upstream of the MS2 stem loops by Gibson assembly (New England Biolabs). Primers used for Xist fragments amplification and cloning are indicated in Supplementary Data 5.

Graindorge A., Pinheiro I., Nawrocka A., Mallory A.C., Tsvetkov P., Gil N., Carolis C., Buchholz F., Ulitsky I., Heard E., Taipale M, & Shkumatava A. (2019). In-cell identification and measurement of RNA-protein interactions. Nature Communications, 10, 5317.

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Measuring MEF Cell Proliferation

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MEF cells were seeded into white 96-well plates at a density of 750 cells/well. The same day, NanoLuc luciferase and substrate (G9711, Promega, Madison, WI, USA) were added simultaneously to the cell culture media. Metabolically active cells can reduce the substrate, which in turn can react with the luciferase. Total luminescence was directly measured using the Infinite 2000 Pro microplate reader (Tecan, Männedorf, Switzerland). After 24 h, the luminescence was measured a second time and the cells were treated with 100 µM H₂O₂. The luminescence was measured again after 24 h and 48 h. Data are expressed as proliferation rate normalized to the first day (proliferation rate = 0).

Wolf C., Zimmermann R., Thaher O., Bueno D., Wüllner V., Schäfer M.K., Albrecht P, & Methner A. (2019). The Charcot–Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress. Cells, 8(10), 1289.

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Quantifying IFN-α Binding Potency

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LIPS experiments were conducted in order to assess the IFN-α-binding potency of S95021. Coding sequences of human IFN-α subtypes without signal peptides were cloned into modified pPK-CMV-F4 fusion vector (PromoCell GmbH, Heidelberg, Germany) downstream of NanoLuc luciferase (Promega, USA), that was cloned into the plasmid instead of Firefly luciferase. HEK 293 cells were transfected with the constructs and secreted NanoLuc-antigen fusion proteins were collected with the supernatants after 48 h. Luciferase immunoprecipitation system (LIPS) assay was adapted from Ref. [32 (link)] and performed in 96-well MultiScreen filter HTS plates (Millipore) at room temperature in 50 mM Tris, pH 7.5 buffer containing 100 mM NaCl, 5 mM MgCl2 and 1% Triton X-100. Serial dilutions of S95021, sifalimumab or rontalizumab were captured onto Protein G Agarose beads (25 μL of 4% suspension, Exalpha Biologicals) and incubated for 1 h with NanoLuc-conjugated IFNs 10⁶ LU per well. After washing, Nano-Glo® Luciferase Assay Reagent was added (Promega, USA) and luminescence intensity measured with Victor X plate reader (Perkin Elmer Life Sciences). Effective concentration 50 (EC₅₀) values were calculated according to the dose-response curves using Prism 8 (GraphPad Software, San Diego, CA).

Duguet F., Ortega-Ferreira C., Fould B., Darville H., Berger S., Chomel A., Leclerc G., Kisand K., Haljasmägi L., Hayday A.C., Desvaux E., Nony E., Moingeon P, & De Ceuninck F. (2021). S95021, a novel selective and pan-neutralizing anti interferon alpha (IFN-α) monoclonal antibody as a candidate treatment for selected autoimmune rheumatic diseases. Journal of Translational Autoimmunity, 4, 100093.

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Quantifying SARS-CoV-2 Antibody Neutralization

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We incubated five-fold serially diluted serum samples from patients who recovered from COVID-19 with SARS-CoV-2 pseudotyped virus for 1 h at 37°C. The mixture was subsequently added to 293TAce2 cl22 cells (for analyses using SARS-CoV-2 Wuhan-Hu-1 [NC_045512] pseudovirus) or HT1080Ace2 cl14 cells (for analyses involving variant pseudovirus panels and the respective Wuhan-Hu-1 Arg683Gly controls).¹⁰ The starting serum dilution on cells was 1:50. We measured NanoLuc luciferase (Promega, Madison, WI, USA) activity in lysates 48 h post-inoculation using the Nano-Glo Luciferase Assay System (Promega) with the Glomax Navigator (Promega). Relative luminescence units were normalised to those derived from cells infected with SARS-CoV-2 pseudotyped virus in the absence of serum. We determined the half-maximal neutralisation titres for serum samples (NT₅₀) using four-parameter non-linear regression using the least squares regression method without weighting.
We considered samples from visit 1 and visit 5 independently, and we evaluated selected cutoff values for each serological assay scale for sensitivity, specificity, and predictive value. More details on the assessment and selection of cutoffs are presented in the appendix (p 2).

Muecksch F., Wise H., Templeton K., Batchelor B., Squires M., McCance K., Jarvis L., Malloy K., Furrie E., Richardson C., MacGuire J., Godber I., Burns A., Mavin S., Zhang F., Schmidt F., Bieniasz P.D., Jenks S, & Hatziioannou T. (2022). Longitudinal variation in SARS-CoV-2 antibody levels and emergence of viral variants: a serological analysis. The Lancet. Microbe, 3(7), e493-e502.

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