Luciferase Overexpression
Luciferase Overexpression - Related Videos
12:55
High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
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Cited by 45 •
2014
We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.
02:01
Assessing Pseudovirus Infection Using Luciferase Reporter Assay
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2026
Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021).This video demonstrates a luciferase-based assay to quantify pseudovirus infection in epithelial cells. The pseudovirus encodes the luciferase gene, which is expressed in infected cells. After washing, and cells are incubated with lysis buffer at an ultra-low temperature, and then thawed to induce lysis.
05:16
Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression
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2025
Source: Bennett, S. A., et al., Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models. J. Vis. Exp. (2019).This video demonstrates the procedure of assessing histone modifications in yeast cells overexpressing neurodegenerative proteins. The steps include cell lysis, centrifugation, electrophoresis, membrane transfer, and antibody-based detection of modified histones.
08:13
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
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2025
This article describes a methodology for overexpressing recombinant Nsp15, a toxic nuclease, in a C41(DE3) expression system, followed by purification of the tagged protein utilizing affinity and size exclusion chromatography. These protocols can be adapted for other challenging toxic proteins.
08:12
Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes
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Cited by 5 •
2011
We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.
FAQs
The promoter sets the production pattern for the engineered enzyme. A strong promoter is suited to generating unusually high luciferase levels, whereas an inducible promoter provides a way to link production to a selected experimental condition. This choice affects whether the readout emphasizes robust signal generation or regulation that changes during the study.
Light is produced when luciferase catalyzes oxidation of its specific substrate. The reaction converts enzyme activity into bioluminescence that can be measured, making the chemical step the bridge between molecular regulation and an experimental signal. Researchers can therefore connect changes in light output with expression, pathway activity, or cellular behavior.
Bioluminescence intensity can serve as a quantitative indicator, but its biological meaning depends on the process being monitored. In one experiment it may report gene expression; in another, pathway activity or cell behavior. Linking the luciferase construct to the relevant regulatory element or cellular process determines how the measured signal should be interpreted.
Researchers first introduce a luciferase-encoding gene into cells and place it under a strong or inducible promoter. After the cells produce the enzyme, they provide the relevant substrate and measure the emitted light. The resulting signal is then compared with the biological activity being studied, such as expression, regulation, or cellular response.
This approach is particularly useful when a study needs a sensitive readout of regulation or response. Reporter assays can examine promoter and enhancer activity, while drug-screening experiments can track changes associated with treatment. In living systems, the same light-based strategy supports noninvasive imaging of cellular processes and observation of cell behavior.
For imaging, light emission provides a readout from cells while they remain in a living system, allowing cellular processes to be assessed noninvasively. The signal can be associated with gene expression, pathway activity, or cell behavior, so the method is useful when researchers need to monitor biological changes in their cellular context.