Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Reporter systems are valuable tools to monitor the expression, interaction and localization of proteins in a cell. Many systems are available—ranging from radiometric and colorimetric readouts to antibody epitope tags and growth inhibition. Yet arguably among the most accessible ways to assay across different cellular proteins, pathways and regulatory events, as well as different experimental conditions, are those based on light emission. Here we sample some of the best and brightest fluorescent and luminescent reporter assay systems available to researchers.
Reporting what?
In a typical reporter system, a transiently or stably introduced genetic construct encodes an element designed to allow visualization when a protein has been expressed. Presence of the protein itself may be the report—such as when expression of green fluorescent protein (GFP) indicates a successful transduction of a virus encoding both it and a gene of interest (GOI), for example, or cotransfection of plasmids separately encoding GFP and the GOI.
Yet such systems are used do far more than that. Reporter expression can be driven by particular chosen or unknown promoters and other regulatory elements, for example, indicating transcription and therefore the presence and activity of cis- or trans-acting factors. A reporter tag can be attached to a GOI, allowing the resultant tagged protein to be detected in an ELISA, Western blot assay or even in vivo. Or the tagged protein in a cell can be imaged across both space and time, perhaps as it interacts with other tagged proteins. Such measurements are commonly used to query the sequelae of genetic alteration, drug treatment, environmental changes and other stimuli.
Luminescent and fluorescent reporters have differing, but increasingly overlapping, roles to play in the research lab. “Typically transcription, translation or protein stability studies utilize luciferase reporters, whereas ligand binding or protein localization, or cell number measurements, are done by fluorescence,” notes George Tzertzinis, senior scientist at New England BioLabs.
As a rule of thumb, fluorescent assays excel for multiplexing and determination of spatial resolution. The higher dynamic range and sensitivity of luminescent reporters make it well suited for gene-expression assays. Put another way, “If you want to follow your protein or are looking at a complex cellular process, I’d recommend a fluorescent reporter,” says Brian Almond, senior product manager at Thermo Fisher Scientific. “However, if you’re looking to know if a gene is turned on or off, then definitely luminescence would be the way you would want to go.”
Fluorescent proteins
The term GFP is often used generically to refer to fluorescent reporter tags or stand-alone proteins. When properly expressed and folded, such sequences absorb light at one wavelength and emit at a longer wavelength—without the need for cofactors or substrates—making them ideal for a variety of imaging and flow cytometric applications. As researchers’ needs are evolving, engineered derivatives of the original GFP and other naturally occurring fluorescent-emitting constructs are being created by many tool providers. Therefore, a host of both emission and excitation wavelengths are available, allowing multiple proteins to be simultaneously imaged with the appropriate equipment. Clontech’s Selection Guide, for example, allows customers to choose from green, yellow, orange, red, far-red and even switchable fluorescent proteins, with one to eight options in each color category.
Compared with the original wild-type form of GFP, many of the newer constructs fold (and therefore are visible) much more rapidly, have increased stability, show decreased toxicity, are significantly dimmer or brighter or even change color over time. They can be made to detect calcium flux or protein-protein interactions. But it’s the same scaffolds being used to do new tricks, says Almond. “A majority of researchers use GFP as a transfection control, to see whether or not their transfection worked.”
Go with the glow
The light given off in luminescence reporter assays is a by-product of the oxidation of a substrate by a luciferase enzyme, often in conjunction with cofactors. And unlike fluorescence, luciferases can turn over multiple molecules of substrate per molecule of enzyme, amplifying the signal many fold before the endpoint reaction is complete. This enables “a much broader dynamic range spanning several orders of magnitude,” notes Tzertzinis.
Deciding on which luminescence reporter is “a little trickier; small differences in reporters can have large effects on results” explains Almond. There are many different enzymes and substrates to choose from, and what works well in one system may not work well in another. For example, firefly luciferase requires a luciferin-based substrate, but Renilla (sea pansy) uses Coelenterazine. Beyond that, substrates may be available to facilitate specific techniques: They may come on more quickly or degrade more quickly, for example, or promise higher fidelity. Most of these choices are the products of innovation from naturally occurring enzymes and substrates to highly evolved reagents. “The original firefly luciferase chemistry was something that you had to measure almost immediately—within a few seconds you had dramatic signal decline, and it was very difficult for the user to get consistent results. We innovated on how to extend the length of the signal,” says Kevin Kopish, senior global strategy marketing manager at Promega. The difference between Promega’s Steady-Glo®, Bright-Glo™ and ONE-Glo™ “is really just signal duration, and this kind of trade-off with overall brightness.”
One trend in the field is to move away from such trade-offs toward smaller, brighter, more stable proteins. Promega’s NanoLuc® (at 19 kD) and Thermo Fisher Scientific’s TurboLuc™ (at 16 kD), for example, are less likely than their larger, dimmer predecessors to engender off-target effects. Another trend is multiplexing of two luminescence assays by using two luciferase reporters and their respective non-cross-reacting substrates, added serially, in the same assay. Several manufacturers offer kits, and researchers can also create such assays as a homebrew.
Several vendors also offer secreted luciferases, obviating the need to lyse the cells to assay for activity.
The well-lit path
With its bright, diminutive enzyme, Promega is “starting to break down the barrier of being able to image a luciferase. That’s still in its infancy, but we’re approaching all the types of applications where you want to start interrogating at the protein level,” says Kopish. And both fluorescence (especially using confocal or multiphoton microscopy) and luminescence are currently used for in vivo imaging of small animals. But for the most part, the two techniques—each rich with its own tools—occupy separate niches, at least for now.