It’s a question I’m asked probably once a week. “What wavelength do I select on my luminometer when performing a luciferase assay?” The question is a good and not altogether unexpected one, especially for those new to bioluminescent assays. The answer is that in most cases, you don’t and in fact shouldn’t select a wavelength (the exception to this rule is if you’re measuring light emitted in two simultaneous luciferase reactions). To understand why requires a bit of an explanation of absorbance, fluorescence, and luminescence assays, and the differences among them.
Absorbance, fluorescence, and luminescence assays are all means to quantify something of interest, be that a genetic reporter, cell viability, cytotoxicity, apoptosis, or other markers. In principle, they are all similar. For example, a genetic reporter assay is an indicator of gene expression. The promoter of a gene of interest can be cloned upstream of a reporter such as β-galactosidase, GFP, or firefly luciferase. The amount of each of these reporters that is transcribed into mRNA and translated into protein by the cell is indicative of the endogenous expression of the gene of interest. Continue reading “Why You Don’t Need to Select a Wavelength for a Luciferase Assay”
Today’s blog was written by guest blogger Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.
Reporter gene assays have been critical for the study of a wide-range of biological questions, from regulation of gene expression to cellular signaling. While reporter gene assays constitute a large group of technologies, here we highlight the diversity of new discoveries enabled by highly quantitative and easily measured bioluminescent luciferase-based reporter assays. Below are our top picks of exciting research discoveries involving the Dual-Luciferase Reporter Assay format using firefly and Renilla luciferases. Continue reading “From Drug Screening to Agriculture to Cardiac Development, Dual Luciferase Reporters Bring You the Story”
Dual-Reporter Assays give scientists the ability to simultaneously measure two reporter enzymes within a single sample. In dual assays, the activity of an experimental reporter is correlated with the effect of specific experimental conditions, while the activity of a control reporter relays the baseline response, providing an essential internal control that reduces variability caused by differences in cell viability or transfection efficiency. The Nano-Glo® Dual-Luciferase® Reporter (NanoDLR™) Assay provides a choice of two sensitive reporters (firefly and NanoLuc luciferases) for use in dual-assay format. Both reporters give state-of-the-art functionality, raising the question “Which luciferase should be the primary reporter and which should be the control?”
This infographic outlines the various NanoDLR dual-reporter assay choices and the situations where you would choose one format over another. Continue reading “Choosing Primary and Control Reporters for Dual-Luciferase Assays”
Helping scientists design experiments and interpret data is what we do best at Promega Technical Services. This may mean spending time at the bench attempting to reproduce anomalous results or forming a team, perhaps with members of other departments, to brainstorm seemingly intractable experimental road blocks. Still, for many of us nothing surpasses the experience of meeting these same scientists face to face whether it be on their home turf or at a booth during a tradeshow. Continue reading “Running A Victory Lap For Promega’s Bioluminescence Technologies”
As part of my job I occasionally search the literature for papers citing use of Promega products in new or interesting ways. Any search on dual-luciferase reporters usually generates a lot of returns. A search for dual-luciferase on Highwire press generates over 700 articles from 2009 alone. So why are dual-luciferase reporter assays so widely used? Continue reading “Why Two Reporters are Better than One”
A IRES (internal ribosome entry site) is a nucleotide sequence that allows for the translation initiation in the middle of a message RNA sequence as part of the greater process of protein synthesis. Typically an IRES is located in the 5’ UTR of RNA viruses and enables translation of the RNAs in a cap-independent manner. (1,2)
Evaluating a particular RNA sequence for IRES activity relies on designing a bicistronic report construct. When an IRES segment is located between two reporter gene (e.g., Chloramphenicol acetyltransferase [CAT] and Luciferase) open reading frames, it can drive translation of the downstream protein coding region independently from the 5´ cap structure bound to the 5´ end of the mRNA (3,4,5). Continue reading “Characterizing IRES Elements using Cell-Based and Cell-Free Expression Systems”
During my childhood, my family and I spent many a vacation in the Swiss Alps. From the mountain tops I used to look out into the horizon as far as the eye could see with peak upon peak stretching out into the distance. If I was lucky, I would have a map that allowed me to identify each peak, perhaps even distinguish the highest from the lowest and thus really get a sense that I understood the underlying topography. However, I quickly realized how little I actually knew about the vast, undulating Swiss countryside. What I had initially observed as a homogenous ‘mat’ of peaks stretching out into the horizon was in fact a rippling of deep valleys that would make an afternoon hike anything but a walk in the breeze.
Looking back on these experiences I am struck by how closely they reflect the landscape of modern science— a broad mat of detailed knowledge with its own peaks of specialization. I am reminded of the words of writer Bill Bryson who described science as “tens of thousands of people that do tiny, tiny things that all accrete into a larger body of knowledge” (1). Continue reading “STATs and ChIPs- Learning A Lesson Or Two About Transcriptional Activation”