Previously, we described some of the advantages of using dual-reporter assays (such as the Dual-Luciferase®, Dual-Glo® Luciferase and the Nano-Glo® Dual-Luciferase® Systems). Another post describes how to choose the best dual-reporter assay for your experiments. For an overview of luciferase-based reporter gene assays, see this short video:
These assays are relatively easy to understand in principle. Use a primary and secondary reporter vector transiently transfected into your favorite mammalian cell line. The primary reporter is commonly used as a marker for a gene, promoter, or response element of interest. The secondary reporter drives a steady level of expression of a different marker. We can use that second marker to normalize the changes in expression of the primary under the assumption that the secondary marker is unaffected by what is being experimentally manipulated.
While there are many advantages to dual-reporter assays, they require careful planning to avoid common pitfalls. Here’s what you can do to avoid repeating some of the common mistakes we see with new users:
Continue reading “Tips for Successful Dual-Reporter Assays”
I’ve got a set of experiments planned that, if all goes well, will provide me with the answer I have been seeking for months. Plus, my supervisor is eagerly awaiting the results because she needs the data for a grant application, so I don’t want to mess it up. However, I am faced with a choice for my firefly and Renilla luciferase reporter assays: Do I use the Dual-Luciferase® Reporter Assay System or Dual-Glo® Luciferase Assay System? What’s the difference? How do I decide which to use? I’m so confused! Help!
Sound familiar? Not to worry! The choice is not difficult once you know how these assays work and how they differ.
Continue reading “Dual-Luciferase or Dual-Glo Luciferase Assay System? Which one should I choose for my reporter assays?”
Even those of us with the greenest thumbs are baffled by the idea of growing food in Antarctica. From my tiny desk plant to my neighbor’s cabbage patch, plants generally have the same requirements: soil, sun and water. At the southern end of the planet, however, those are all scarce commodities. Nonetheless, on April 5, 2018, the team managing the EDEN-ISS greenhouse at Neumayer III announced that they had harvested 8 pounds of salad greens, 18 cucumbers and 70 radishes. This project has implications beyond just Antarctica, from moderate climates on Earth to future Mars missions. Continue reading “From Antarctica to Mars: Growing Food in Extreme Conditions”
Genetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).
Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation
However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.
At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?
To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.
So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.