When should I use fluorescent proteins, luciferases or LacZ?
Fluorescent proteins, luciferases, and LacZ are all recombinant protein-based reporters that can be used for localization or imaging studies. However, these systems differ in several important ways that determine their suitability for different experimental designs.
Fluorescent proteins and luciferases are both types of proteins which emit light, which is then detectable with a camera or similar device. Fluorescent proteins function by absorbing light of one color (excitation), and then emitting lower-energy light of a different color (emission). In contrast, luciferase (and other bioluminescence enzymes) generate light by catalyzing a chemical reaction in which a substrate (i.e. luciferin) is oxidized, emitting photons as a reaction product.
Fluorescent proteins are much brighter than luciferase, which benefits many types of experiments, but in tissue samples or live animals, background, autofluorescence, and light scattering can make fluorescent proteins problematic. Since luciferase activity requires luciferin, this substrate must be added to culture media or injected into live animals prior to analysis. In many cases this can limit reproducibility or quantitation of experiments, when compared to fluorescent proteins.
Unlike the above two types of markers, LacZ does not emit light. Instead, the LacZ gene product, β-galactosidase, can catalyze the conversion of X-gal into an opaque blue compound similar to indigo. Therefore, in order to visualize LacZ, samples must first be stained with X-gal solution, and this staining procedure is not compatible with live samples. Although highly sensitive, LacZ/X-gal staining is not quantitative, and does not provide the high resolution possible with fluorescent or bioluminescent proteins.
In general, we recommend LacZ for gene expression studies in whole-mount embryos or tissue sections. For live animal studies, either fluorescence or bioluminescence may be a good choice, depending on a number of factors, such as whether imaging deep inside tissues is necessary and whether marker brightness may be an issue.