Firefly luciferase assay

Manufactured by Promega

Sourced in United States

The Firefly luciferase assay is a laboratory tool that measures the enzymatic activity of firefly luciferase, a bioluminescent protein. This assay is used to quantify gene expression or protein levels in a variety of biological samples.

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Lab products found in correlation

Prism v5, by GraphPad (1 mentions) Centro lb 960 luminometer, by Berthold Technologies (1 mentions) Reporter lysis buffer, by Promega (1 mentions) β galactosidase, by Promega (1 mentions)

Close spelling variants of this product

Luciferase assay (182 citations) Firefly luciferase assay system (43 citations) Firefly luciferase (31 citations) Firefly Luciferase Assay Kit (29 citations) Firefly/Renilla Dual-Luciferase Reporter Assay System (6 citations) Firefly and Renilla Dual-Glo™ Luciferase Assay System (6 citations) Firefly luciferase assay substrate (4 citations) Firefly luciferase assay reagent (2 citations) Firefly Luciferase Reporter Assay System (2 citations)

5 protocols using firefly luciferase assay

NF-κB and AP-1 Activation Assay

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RAW 264.7 cells were transfected with NFκB or AP-1 plasmid and TK-Renilla using Solfect™ (Biosolyx, Daegu, Republic of Korea) according to the manufacturer's instructions. Cells were then plated in 24-well plates at a density of 3 × 10⁵ cells/mL. After 24 h, the cells were treated with the DIE extract followed by 0.1 μg/mL LPS 30 min later. Cells were incubated for 5 h and the expression of NFκB and AP-1 was measured using the Promega firefly luciferase assay (Madison, WI, USA) according to the manufacturer's instructions. Luciferase activity was normalized to that of TK-Renilla luciferase.

Lee Y.Y., Yuk H.J., Saba E., Kim S.D., Kim D.S., Kopalli S.R., Oh J.W, & Rhee M.H. (2022). Duchesnea indica Extract Ameliorates LPS-Induced Septic Shock in Mice. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 5783867.

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Neutralizing Antibody Titers Against SARS-CoV-2 Variants

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Serial three-fold serum dilutions starting at 1:40 were incubated 30 min at 4 °C with B.1, Omicron BA.1 or Omicron BA.2 Spike pseudotypes. The serum/pseudotype mixture was then added to HEK293T-ACE2-TMPRSS2 cells (5 × 10⁴ cells/well) in DMEM containing 10% FBS for 60 h. Cells were then lysed using the Promega lysis buffer (E1531 from Promega, Belgium) and a freeze-thaw cycle. Then, Firefly Luciferase activity was assessed using the Firefly Luciferase Assay, following the manufacturer’s recommendations (E4550 from Promega, Belgium). Percent infection was calculated relative to infected cells in the absence of test serum. The IC50 was determined by inferring the 4-parameter nonlinear regression curve fit with unconstrained top and bottom values using GraphPad Prism v5 and was log-transformed into 50% neutralizing titer (NT50) as above.

Santos da Silva E., Servais J.Y., Kohnen M., Arendt V., Gilson G., Staub T., Seguin-Devaux C, & Perez-Bercoff D. (2023). Vaccine- and Breakthrough Infection-Elicited Pre-Omicron Immunity More Effectively Neutralizes Omicron BA.1, BA.2, BA.4 and BA.5 Than Pre-Omicron Infection Alone. Current Issues in Molecular Biology, 45(2), 1741-1761.

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Luciferase Assay for Transcriptional Regulation

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For luciferase assay, 1.2 × 10⁵ A549 and 10⁵ Nthy cells per well were plated in 24-well plates. After 24 h, A549 cells were transfected with 400 ng lung reporter plasmid (SP-A2, SP-B, SP-C, or ABCA3 luciferase), and 400 ng wild-type or mutant NKX2-1 plasmid. Nthy cells were transfected with 200 ng thyroglobulin reporter plasmid, 200 ng PAX8 plasmid and 200 ng wild-type or mutant NKX2-1 plasmid. The total amount of plasmids was kept constant by the addition of the respective empty vector (pcDNA3-1). After 24 h, cells were harvested, and underwent firefly luciferase assay (Promega) in a TriStar system (Berthold Technologies GmbH & Co. KG, Germany). The promoter activation was calculated after normalization to the total luminescence of the experiment, in five independent experiments, each condition performed in duplicate.

Delestrain C., Aissat A., Nattes E., Gibertini I., Lacroze V., Simon S., Decrouy X., de Becdelièvre A., Fanen P, & Epaud R. (2023). Deciphering an isolated lung phenotype of NKX2-1 frameshift pathogenic variant. Frontiers in Pediatrics, 10, 978598.

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HTLV-1 Infectivity Assay in Jurkat Cells

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HEK293FT cells were seeded in 6 wells of a 12-well plate at 3 × 10⁵ cells/mL/well. After 24 h, HTLV-1 infectious plasmid (pX1-MT-M) or pME-18S as negative control was introduced to 3 wells each at 1 μg/well by PEI. After 48 h, the cells were co-cultured with 1 × 10⁵ cells/well of Jurkat cells containing HTLV-1-LTR driven firefly luciferase gene. After 24 h, LTR activation in Jurkat cells was detected by firefly luciferase assay (Promega, Corp., Madison, WI, USA) on Centro LB 960 luminometer (Berthold Technologies GmbH & Co KG, Bad Wildbad, Germany) as the amount of infectious virus particles. The protein level of the cell lysate in each well was measured, and the corresponding firefly activity was normalized by the protein level (mg protein).

Nakano K., Yokoyama K., Shin S., Uchida K., Tsuji K., Tanaka M., Uchimaru K, & Watanabe T. (2022). Exploring New Functional Aspects of HTLV-1 RNA-Binding Protein Rex: How Does Rex Control Viral Replication?. Viruses, 14(2), 407.

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Luciferase and β-Galactosidase Assay Protocol

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Luciferase and β-galactosidase assays were carried out according to the manufacturer’s protocol (Promega). Briefly, for luciferase assays, cells were washed with 1X PBS buffer containing 137-mM NaCl, 2.7-mM KCl, 8.1-mM Na₂HPO₄, and 1.47-mM KH₂PO₄ and incubated with Reporter Lysis Buffer (Promega) for 15 min. Cells were collected and subjected to one rapid freeze/thaw cycle, and then immediately used for the firefly luciferase assay (Promega). The relative luciferase activities of the samples were counted with a luminometer at a 2-s measurement delay and 10-s measurement read in the presence of luciferin substrate.
For β-galactosidase assays, the cell extracts were incubated in a buffer containing 1.33 mg/mL of the substrate ONPG (O-nitrophenyl-D-galactopyranoside), 200-mM sodium phosphate buffer, 2-mM MgCl₂, and 100-mM β-mercaptoethanol, at 37 °C for 30 min. The reaction was stopped with 1-M sodium carbonate and the absorbance was determined by spectroscopy at 420 nm.

Ye L., Gu L., Caprioli J, & Piri N. (2018). RNA-binding protein Rbpms is represented in human retinas by isoforms A and C and its transcriptional regulation involves Sp1-binding site. Molecular genetics and genomics : MGG, 293(4), 819-830.

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