Robert Hooke first coined the term “cell” after observing plant cell walls through a light microscope—little empty chambers, fixed in time and space. However, cells are anything but fixed.
Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.
Visualizing and measuring these fluid interactions at the level of the cell without perturbing them is the goal of every cell biologist.
A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal. Continue reading “If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein Interactions”
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.