Luciferase assay reagent

Manufactured by Promega

Sourced in United States, Japan, Germany, Canada, Spain, China, France, Switzerland

Luciferase assay reagent is a laboratory tool used to measure the activity of the luciferase enzyme. It provides the necessary components to facilitate the luciferase-catalyzed oxidation of luciferin, resulting in the emission of light that can be quantified to determine the presence and levels of luciferase in a sample.

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Lab products found in correlation

Passive lysis buffer (plb), by Promega (59 mentions) Reporter lysis buffer, by Promega (53 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (32 mentions) Cell culture lysis reagent, by Promega (24 mentions) Lysis buffer, by Promega (14 mentions) Luciferase assay system, by Promega (13 mentions) Glomax 96 microplate luminometer, by Promega (12 mentions) Luciferase cell culture lysis reagent, by Promega (11 mentions) Luminescence counter, by Promega (9 mentions) Glo lysis buffer, by Promega (9 mentions) Glomax 20 20 luminometer, by Promega (9 mentions) Dry thermo unit, by TAITEC (8 mentions) Fluostar optima, by BMG Labtech (8 mentions) Glomax multi detection system, by Promega (8 mentions) Tnt t7 quick coupled transcription translation system, by Promega (7 mentions) Prism software, by GraphPad (6 mentions) Infinite m200 pro, by Tecan (6 mentions) Dual luciferase reporter assay system, by Promega (6 mentions) Glomax dual luminometer, by Promega (6 mentions) Pgl4.15 vector, by Promega (5 mentions)

Spelling variants of this product

Luciferase assay (182 citations) Luciferase Assay Reagent II (55 citations) Luciferase reagent (50 citations) Dual-Luciferase Reporter Assay reagent (30 citations) Dual-Luciferase Reporter Assay System reagents (11 citations) Light switch luciferase assay reagents (5 citations) Dual-Glo Luciferase Assay System reagents (5 citations) Luciferase reporter gene assay reagents (2 citations) Dual-luciferase reporter (DLR) assay kit reagents (2 citations) Duo-Luciferase reporter assay reagents (2 citations)

446 protocols using luciferase assay reagent

Luciferase Reporters for β-Catenin and NF-κB Activity

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The luciferase reporter for assessing β-catenin activity, which contains TCF/LEF sites was a gift from Dr Randall Moon (Addgene plasmid #12456)19 (link). The reporter cassette containing NF-κB-binding sites was obtained from Clontech. Constructs were cloned into the pAd-DEST Gateway vector (Invitrogen), to produce adenoviruses expressing these reporter genes.
For luciferase assay, 7 × 10⁵ NRCMs were plated in 24-well plates in 1 ml maintenance media for 24 h. Next, the media was changed with either maintenance media or fibroblasts conditioned media. Adenovirus-encoding the TCF/LEF-luciferase construct was added and cells were then stimulated with 30 μM phenylephrine for 72 h. Luciferase assays were performed using luciferase assay reagent (Promega) following manufacturer's recommended protocol.
The NF-κB-luciferase reporter was used to examine NF-κB activity in cardiac fibroblasts. In brief, 5 × 10⁴ ACFs were plated in 24-well plates for 24 h. Next, the cells were infected with the NF-κB reporter adenovirus and incubated for 72 h at 37 °C. Luciferase assays were performed using luciferase assay reagent (Promega) following the manufacturer's recommended protocol.

Mohamed T.M., Abou-Leisa R., Stafford N., Maqsood A., Zi M., Prehar S., Baudoin-Stanley F., Wang X., Neyses L., Cartwright E.J, & Oceandy D. (2016). The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy. Nature Communications, 7, 11074.

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Measuring SARS-CoV-2 Pseudovirus Inhibition

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The infectivity of SARS-CoV-2, SARS-CoV, and vesicular stomatitis virus (VSV) for 293T cells or 293T cells stably expressing human ACE2 (293T/ACE2) was determined by a single-cycle infection assay as described previously (62 (link)). To produce pseudoviruses, 293T cells were cotransfected with a backbone plasmid (pNL4-3.luc.RE) that encodes an Env-defective, luciferase reporter-expressing HIV-1 genome and a plasmid expressing the S protein of SARS-CoV-2 or SARS-CoV or the G protein of VSV. Cell culture supernatants containing the released virions were harvested at 48 h posttransfection, filtrated, and stored at −80°C. To measure the inhibitory activity of peptide inhibitors, pseudoviruses were mixed with an equal volume of a serially 3-fold-diluted peptide and incubated at 37°C for 30 min. The mixture was then added to 293T/ACE2 cells at a density of 10⁴ cells/100 μl per plate well. After they were cultured at 37°C for 48 h, the cells were harvested and lysed in reporter lysis buffer, and luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega, Madison, WI, USA). The 50% inhibitory concentration (IC₅₀) was calculated as the final cell culture concentration of an inhibitor that caused a 50% reduction in relative luminescence units (RLU) compared to the level of the virus control subtracted from that of the cell control.

Zhu Y., Yu D., Yan H., Chong H, & He Y. (2020). Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity. Journal of Virology, 94(14), e00635-20.

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Ikaros Modulates IL7R and SH2B3 Promoters

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The pGL4.15 luciferase reporters construct for the IL7R and SH2B3 promoters were constructed by insert of the IL7R promoter (−1000bp to 0bp) and SH2B3 promoter (−100 to +300bp). Transient luciferase assays were performed in HEK293T cells using Promega luciferase assay reagents and measured with a luminometer following the manufacturer's instructions. Luciferase activity was calculated as fold change relative to values obtained from pGL vector only control cells and expressed as a percentage of pcDNA 3.1-Ikaros transfection-induced luciferase activity versus that of pcDNA3.1 vector alone. All transfection and reporter assays were performed independently in triplicate at least three times.

Ge Z., Gu Y., Xiao L., Han Q., Li J., Chen B., Yu J., Kawasawa Y.I., Payne K.J., Dovat S, & Song C. (2016). Co-existence of IL7R high and SH2B3 low expression distinguishes a novel high-risk acute lymphoblastic leukemia with Ikaros dysfunction. Oncotarget, 7(29), 46014-46027.

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CpG-ODNs Immunomodulatory Protocol

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CpG-oligodeoxynucleotides (CpG-ODNs) were purchased from Invitrogen (Carlsbad, CA, USA) or Genomics BioSci & Tech (New Taipei, Taiwan). Anti-FLAG® antibody, Tricaine, and all of the chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), while anti-actin antibody was purchased from Santa Cruz Biotech Inc. (Dallas, TX, USA). Enzyme-linked immunosorbent assay (ELISA) kits for the detection of human cytokines were purchased from eBioscience (San Diego, CA, USA), and luciferase assay reagents were purchased from Promega (Madison, WI, USA).

Yeh D.W., Lai C.Y., Liu Y.L., Lu C.H., Tseng P.H., Yuh C.H., Yu G.Y., Liu S.J., Leng C.H, & Chuang T.H. (2017). CpG-oligodeoxynucleotides developed for grouper toll-like receptor (TLR) 21s effectively activate mouse and human TLR9s mediated immune responses. Scientific Reports, 7, 17297.

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Measuring HIV-1 Env Infectious Ability

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The infectious ability mediated by HIV-1 recombinant Env protein was measured by a single-cycle infection assay as described previously [20 ]. Briefly, HIV-1 pseudovirus was generated by co-transfecting 293 T cells with an Env-expressing plasmid and a backbone plasmid pSG3^Δenv. The supernatant was harvested 48 h after transfection, and 50% tissue culture infectious dose (TCID₅₀) was determined using TZM-bl cells (NIH AIDS Reagent Program). The infectious ability of recombinant strains was determined by infection of TZM-bl cells with 100 TCID₅₀ virus dose. The cells were incubated for 48 h at 37 °C, and the luciferase activity (relative light unit, RLU) was measured using luciferase assay reagents (Cat No.: G7941, Promega) and a luminescence counter GloMax (Promega). Student t test was performed to compared the difference. p value less than 0.05 was considered significant statistical difference.

Wei H., Yu D., Geng X, & He Y. (2020). Defective HIV-1 envelope gene promotes the evolution of the infectious strain through recombination in vitro. BMC Infectious Diseases, 20, 569.

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Transcriptional Regulation of BCL6 and BACH2

Cited 2 times

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Promoters of BCL6 (-2000bp to +100bp) and BACH2 (-1000bp to +200bp) were cloned into pGL4.15 vector (Promega, Madison, WI, USA). The transient luciferase assay was performed in HEK293T cells using the Promega luciferase assay reagents and measured with a luminometer according to the manufacturer instructions [33 (link), 35 (link), 48 (link)]. The firefly luciferase activities were calculated as fold-change relative to values obtained from pGL4.15 vector only control cells, and expressed as a percent of pcDNA3.1-Ikaros transfection-induced luciferase activity vs. the pcDNA3.1 vector. All transfection and reporter assays were performed independently with ≥3 replicates.

Ge Z., Zhou X., Gu Y., Han Q., Li J., Chen B., Ge Q., Dovat E., Payne J.L., Sun T., Song C, & Dovat S. (2016). Ikaros regulation of the BCL6/BACH2 axis and its clinical relevance in acute lymphoblastic leukemia. Oncotarget, 8(5), 8022-8034.

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Radiation-Induced Transcriptional Regulation

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MLE12 cells were cultured in 60-mm plates using complete medium until they reached ~70% confluency, and were then co-transfected with either SBE-luciferase + TK-Renilla luciferase or NF-κB-luciferase + TK-Renilla luciferase. After 24 h, the cells were irradiated using an image-guided small-animal irradiator, and the ratio of firefly/Renilla luciferase was calculated and normalized to untreated controls. Luciferase activity was measured using luciferase assay reagents (Promega Corp., Madison, Wi, USA).

Kim H., Park S.H., Han S.Y., Lee Y.S., Cho J, & Kim J.M. (2020). LXA4-FPR2 signaling regulates radiation-induced pulmonary fibrosis via crosstalk with TGF-β/Smad signaling. Cell Death & Disease, 11(8), 653.

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Pseudovirus Infection Inhibition Assay

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Inhibitory activity of fusion inhibitors on diverse pseudoviruses was determined by a single-cycle infection assay as described previously [6 (link)]. In brief, pseudovirus for HIV-1, SIV, H5N1, H7N9, RSV, or VSV was generated by cotransfecting HEK293 T cells with an Env-encoding plasmid and a backbone plasmid (pNL4-3.luc.RE) that encodes an Env-defective, luciferase reporter-expressing HIV-1 genome. After transfection 48 h, virus-containing supernatants were harvested and 50% tissue culture infectious dose (TCID₅₀) was measured. Peptides or lipopeptides were prepared in 3-fold dilutions, mixed with 100 TCID₅₀ of viruses. After incubation for 1 h at room temperature, one hundred microliters of the mixture were added to target cells (TZM-bl for HIV-1, SIV and VSV; MDCK for H5N1 and H7N9; HEp-2 for RSV) that were plated at 10⁴ cells/well and then incubated for 48 h at 37°C. The cells were harvested and lysed in reporter lysis buffer, and luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega), and IC₅₀ values were calculated as described above.

Yu D., Zhu Y., Yan H., Wu T., Chong H, & He Y. (2021). Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus. Emerging Microbes & Infections, 10(1), 810-821.

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Measuring HIV-1 Entry Inhibition

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HIV-1 entry was determined by a single-cycle infection assay as described previously [30 (link)]. Briefly, Env-based pseudoviruses were generated by cotransfecting HEK293T cells with an Env-expressing plasmid and a backbone plasmid pSG3^ΔENV that has an Env-defective, luciferase-expressing HIV-1 genome. Virus supernatants were harvested 48 h after transfection, and 50% tissue culture infectious doses (TCID₅₀) were determined in TZM-bl cells. To evaluate the infectivity of pseudoviruses, the same amount of viral particles was normalized by p24 antigen. To measure the antiviral activity of fusion inhibitors, peptides were prepared in graded concentrations, mixed with 100 TCID₅₀ of viruses, and then incubated 1 h at room temperature. The mixture was added to TZM-bl cells (10⁴/well) and then incubated 48 h at 37 ℃. Luciferase activity was measured using luciferase assay reagents and a luminescence counter (Promega, Madison, WI, USA). Percent inhibition of the pseudovirus by an inhibitor and 50% inhibitory concentration (IC₅₀) were calculated using GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA).

Geng X., Liu Z., Yu D., Qin B., Zhu Y., Cui S., Chong H, & He Y. (2019). Conserved Residue Asn-145 in the C-Terminal Heptad Repeat Region of HIV-1 gp41 is Critical for Viral Fusion and Regulates the Antiviral Activity of Fusion Inhibitors. Viruses, 11(7), 609.

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Dual Luciferase Assay in RAW 264.7 Cells

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For dual luciferase assay, RAW 264.7 cells were grown in a 96-well plate, and then cells were cotransduced with a pCignal Lenti-ARE reporter (Qiagen, Hilden, Germany) and pCignal Lenti-TK-Renilla (Qiagen, Hilden, Germany). After 72 h, cells were treated with 10 μM 4-PG for 8 h. Treated cells were lysed with passive lysis buffer (Promega, Fitchburg, WI, USA) and mixed with luciferase assay reagents (Promega). The chemiluminescent signal was detected using a SpectraMax L Microplate reader (Molecular Devices, Sunnyvale, CA). Firefly luciferase was normalized to Renilla luciferase in each sample.

Park J., Chen Y., Zheng M., Ryu J., Cho G.J., Surh Y.J., Sato D., Hamada H., Ryter S.W., Kim U.H., Joe Y, & Chung H.T. (2018). Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1. Oxidative Medicine and Cellular Longevity, 2018, 2747018.

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