Fluorescent Tags: More Than Innocent Bystanders

A fluorescent tag, also known as a label or probe, is a molecule that is chemically attached to a biomolecule such as a protein, antibody, or synthetic compound, in order to aid in labeling and detection. Scientists have been using tags for well over a century as research tools, but here we discuss some of the latest research that demonstrates that tags are not innocent pretty colored labels, but active chemicals that can alter the course of the experiment.

Imagine a scenario where a researcher is working with a small molecule, and he has reason to believe it has potential as an interesting anti-cancer drug. He suspects that the molecule enters cells, then continues on into the nucleus to shut down a detrimental gene. So this researcher makes a new molecule, just like the original one except that it is now attached to a fluorescent label. He places this compound a dish of cells, put the cells under a fluorescent microscope, and voila! The cells’ nuclei light up fluorescent green. The researcher has proven that the original small molecule enters cell nuclei.

Actually…he hasn’t. Fluorescent tags are not just splashes of color that are along for the ride. Like the small molecule, it too has atoms and covalent bonds. Many times, the tag is a hefty percentage of the overall molecular weight. With chemistry of its own, it might influence what a cell decides to do with the molecule, whether to incorporate it or exclude it. In other words, a labeled and an unlabeled molecule may travel quite differently.

A similar pitfall can affect proteins under scrutiny, as well. Proteins are commonly tagged with other, relatively small, proteins, most notably Green Fluorescent Protein. It is ubiquitous in research, earned some of its discoverers/developers a Nobel Prize, and has been a crucial part of many important discoveries.

Such tags were used to visualize bacterial proteins called Clp proteases. Research had revealed that labeled Clp proteases show up as a single spot within a bacterium. That suggested that, unless bacteria managed to divide right in the middle of the speck, only one daughter was the owner of this important spot, and the other got none!

However, a careful investigation unveiled the real story: In a strain in which these proteases were un tagged, researchers found that the Clp protease they were studying was nearly evenly distributed between daughters.¹ It turned out that the fluorescent tag strengthened the protease’s tendency to self-associate. Further studies suggested that this type of confusion is not limited to Clp, as these same researchers demonstrated that five other bacterial proteins that had been determined to form clumps in fact only did so in large part because of their fluorescent tags.

Of course, researchers should not to give up on fluorescent tags. They are truly powerful methods to conduct groundbreaking research. And to provide a wider body of support for your hypotheses, studies using fluorescence tags can be combined with label-free technologies such as surface plasmon resonance, an area of expertise for many CROs listed on Scientist. In order to truly understand the data, researchers need to remember that fluorescent tags often have side effects that are more than just their color.