Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Reporter proteins are like tiny, color-coded, Post-It® tags that attach to, and mark the presence of, molecules of interest. They generally come in two flavors, fluorescent or luminescent (including chemiluminescent and bioluminescent), and they can be used to detect the locations, interactions and activities of both proteins and nucleic acids.
Oftentimes, expression plasmids are available encoding either type of reporter, and someone new to the technology might find the choices confusing. Here are some guidelines to help.
When to choose fluorescent reporters
One of the first considerations in selecting a reporter is your research goal. If you're interested in protein localization in cultured cells, "a clear choice is for a fluorescent reporter,” says Kevin Kopish, senior strategic marketing manager at Promega. “Primarily, fluorescent reporters are used as a means of studying protein dynamics.” Chemiluminescent tools, in contrast, typically are reserved for studies of gene regulation, says Kopish.
Fluorescent reporters also are popular choices for multiplexing. “Multiplexing multiple reporters is possible by choosing compatible fluorescent proteins or by altering the fluorophore used to label the protein,” says Kopish. The ability to multiplex is particularly helpful in some of the common applications of fluorescent reporters, including microscopy, flow cytometry and fluorescence-activated cell sorting. Conversely, chemiluminescent signals work better when probing bulk populations, such as in Western blotting, in vivo imaging and multiwell, plate-based assays.
Because fluorescent reporters tend to be very bright, it is helpful to match brightness with target abundance (using brighter dyes for weaker signals) when multiplexing them, advises Brian Almond, senior product manager in cellular analysis at Thermo Fisher Scientific. Also, be sure the emission wavelengths of the fluorescent reporters are sufficiently distinct, so they can be cleanly resolved. “Several different emission colors can be monitored from the same sample, which results in a content-rich experimental result,” Almond says.
Fluorescent reporters also can be used to assess intermolecular interactions, for instance using fluorescent resonance energy transfer and its many derivatives. In the proximity ligation assay, such as Sigma-Aldrich’s Duolink® assay, fluorescence occurs when the two proteins of interest are in close physical proximity. “It needs to be fluorescent [rather than chemiluminescent], because the microscopy cameras they use [for proximity assays] don’t work well with luminescence,” says Carol Kreader, scientist in applied R&D at Sigma-Aldrich.
When to choose chemiluminescent reporters
Chemiluminescence is a popular choice for gene-expression analyses, says Kopish. One common application, for instance, is pairing a regulated promoter to luciferase and then assessing promoter activity based on light output from protein extracts. Chemiluminescence is mainly used to measure signals from groups of cells (in individual microplate wells, for instance) rather than individual cells. “The use of chemiluminescence, which includes bioluminescence, has typically been restricted to whole-well assays and can output signals over several logs of range,” says Kopish. “These traits also make for quantitative, rather than qualitative, tools.”
Sigma takes advantage of such quantitative luminescent tools in its MISSION® 3’UTR Lenti GoClone™ system (PDF) for microRNA detection, says Kreader. “They have LightSwitch Luciferase fused to the 3’-untranslated region of several thousand different miRNAs. The idea here is that if the miRNA binds, then the luminescence goes down.” The system is used to validate miRNA targets and study post-transcriptional regulation of genes.
Besides quantitation, luminescence has other strengths, including sensitivity and low background intensity. Some biological molecules emit fluorescence upon excitation, Almond notes, a nonspecific emission called autofluorescence that can interfere with fluorescent reporters, he says. Chemiluminescent reporters, he says, "display virtually no background,” and thus are often used when researchers need to amplify a signal or to minimize background signals. “Chemiluminescent reporters are well suited to Western [blot] analysis and ELISA applications,” says Almond.
Because of their lower background, chemiluminescent signals also work well in tissues and with in vivo studies of small animals, for example using whole-animal imagers. “Luminescent reporters have been used very successfully in animals, again made possible by the very sensitive nature of these reporters,” says Kopish. “Fluorescence struggles in animals due to the scattering and absorption of light as well as the possibility of autofluorescence creating a very high background signal.”
Blurring the lines
A recent release from Promega, NanoLuc® luciferase, could change the definitions of what different types of reporters can do. NanoLuc yields higher luciferase activity than other luciferases, so it gives a strong signal. But it also differs from previous luciferases by being physically smaller. “The very high activity is allowing us to blur the lines between what can be accomplished with a luminescent reporter,” says Kopish. “For instance, allowing us to detect fusion proteins using a microscope for localization, but also measure the whole-well population for higher throughput.”
In contrast to the conventional wisdom discussed above, NanoLuc technology should enable researchers to use luminescent reporters to study two proteins simultaneously, says Kopish, and even molecular interactions—assays traditionally reserved for fluorescence. In March 2015, Promega is expected to release a dual-luciferase assay that uses both NanoLuc and firefly luciferase reporters to measure molecular interactions though bioluminescence resonance energy transfer, says Kopish. “With this technology, we can now look at protein interactions, and the sensitivity allows us to keep protein expression at near endogenous levels.”
Yet another application of the new reporter is in gene expression. Promega and Horizon Discovery recently collaborated to incorporate NanoLuc into a CRISPR/Cas9-based genome engineering system. The system enabled them to create reporter gene assays either by replacing an allele with NanoLuc or by inserting NanoLuc to create a fusion protein. “This creates reporter assays that have the correct context in the chromatin and would be influenced by short- and long-range regulators of gene expression,” says Kopish. “For the protein fusions, the expression of the tagged protein is less than the total amount of the protein of interest, so there are no artifacts from overexpression, creating much more relevant assays.”
If you want to create your own relevant assays, the tools clearly are available to help, whichever reporter you choose. All it takes is a little cloning. So what are you waiting for?
Image: iStockPhoto