Laura Lane has worked as a health and science journalist since 1997. She received her master's degree in biology from Stanford University. Since then, she has written for the Dallas Morning News, the Contra Costa Times, Shape magazine, WebMD, Yoga Journal, Diagnostic Imaging, the International Medical News Group, The Scientist, Bio IT World and Biocompare.
In the courtroom, there’s little that can sway the jury more than eyewitness testimony. It’s compelling evidence. Now, advanced protein-labeling technologies are enabling researchers to adopt that same strategy. They can literally peer into live cells and watch labeled proteins going about their business.
Aaron Hoskins has certainly embraced this straightforward approach. An assistant professor of biochemistry at the University of Wisconsin, Madison, Hoskins painstakingly details exactly how proteins of the spliceosome collaborate to splice pre-mRNA transcripts in the correct spots. The work involves visually tracking the activity of one protein, or sometimes a few proteins, in each experiment.
“Imaging allows [us] to see what otherwise would be invisible,” Hoskins explains. It “allows us to ask questions that previously couldn’t be addressed, like splicesome biochemistry.”
Others can ask those questions in their own systems, too, and without too much difficulty. From protein fusions to chemical handles, the protein-labeling toolbox is overflowing with options.
It’s a SNAP!
One common approach to protein labeling fuses the protein of interest with a fluorescent protein. Alternatively, you can tag with a fusion protein that you can label on-demand, such as the SNAP- and CLIP-tags.
Engineering from DNA-repair proteins, SNAP- and CLIP-tags are expressed as fusion proteins with the target protein. The signal comes about when the tag covalently reacts with a substrate carrying a fluorophore, biotin or other probes, which are thus physically linked to the target protein. The SNAP-tag® reacts with benzylguanine- or chloropyrimidine-linked molecules; the CLIP-tag reacts with benzylcytosine.
Unlike proteins fused to green fluorescent protein that constitutively glow, SNAP-tags only light up when you add the substrate-linked probes, explains Chris Provost, product manager at New England Biolabs, which markets SNAP-tag. And, you have the flexibility of using other probes besides fluorophores, such as biotin or magnetic beads. You can even incorporate blocking agents or quenching groups by linking them to benzylguanine.
Hoskins prefers SNAP-tags because they “allow [him] to label really quickly using a minimal amount of fluorophores,” he says, which provides a cost savings. Researchers using labels with slower reactions commonly try to expedite the process by adding excess fluorophores.
Dyes used with SNAP-tags, such as organic dyes, shine brighter than traditional fluorophores, says Justin Taraska, principal investigator at the National Heart, Lung and Blood Institute, who uses protein labels to study endocytosis. In addition, they don’t blink or bleach as much as fluorescent proteins, he says.
Click-clack
Instead of tagging the actual target protein, you may opt to tag its antibody using so-called “Click” chemistry.
Click chemistry hinges on a reaction between azide and alkyne chemical groups. This binding event does not occur in nature, and it forms a covalent linkage. Either group can carry the probe, while the other is bound to the target (in this case, the antibody).
“Customers can have a Click handle on the antibody and react it with the partner with the attached fluorophore and simply go,” says Kathy Free, senior manager of product management at Life Technologies, which markets Click-iT® reagents, emphasizing the convenience of the technology.
The company has harnessed that chemistry to develop the SiteClick™ Antibody Labeling System. The kit provides reagents to attach azide tags to N-acetylglucosamine residues on the heavy chains of IgG antibodies, which aren’t involved with antigen recognition or binding. These azide tags can then bind to probe-carrying alkynes, bringing light to the antibody and thus, to the target protein.
The system is designed for reproducibility, Free says. “It’s always attaching to the same place.”
“SiteClick is enabling in that you can pretty much put any probes on the antibody, maintain the integrity of the antibody and not worry about any of the antigen recognition sites or the functional groups that keep the antibody together,” she says.
Usual and customary expression
Understanding the true behavior of a protein requires that it be expressed at physiological levels. But some researchers like to overexpress the target protein to ensure they can spot it, says Taraska. “You get mistargeting because there’s too much protein floating around.”
One way to achieve physiological levels of expression is to allow the cell to express the protein from its normal genomic location but tagged with a probe.
Sigma Aldrich’s zinc finger nuclease technology helps researchers navigate in that direction. This genome-editing approach consists of a pair of DNA-binding domains targeting your specific gene. Once bound, the accompanying DNA cleavage domains together snip both strands of the DNA. Then, through homologous recombination, one of the cell’s natural repair mechanisms, the cell inserts your sequence of interest, such as a fluorescent protein gene.
By contrast, “If you’re relying on just random integration of a plasmid with your gene, it can go anywhere,” says Erika Holroyd, a product specialist at Sigma Aldrich, explaining the specificity of zinc finger nuclease.
Viral and plasmid delivery of fusion proteins are notorious for overexpression. Users can modulate that by using weaker promoters, Taraska says.
Or, you can control the amount of virus you use to infect cells, says Michael Haugwitz, associate director of research and development in cell biology at Clontech Laboratories. “You can regulate the expression level rather well and adjust it to physiological levels,” Haugwitz says. “By adding fewer viral particles, you can get lower expression.”
True colors
With imaging, the best observations come from maximizing the signal-to-noise ratio. Natural autofluorescence—i.e., noise—glows bluish-green, Taraska says. “You get around it by using red color dyes or really bright dyes, so that the majority of the signal is coming [only] from the dyes,” he says.
New England Biolabs is developing a far-red fluorophore that is also cell permeable. Or, you can look to Clontech for far-red fluorescent proteins such as E2-Crimson and mPlum.
Clontech also provides photoswitchable fluorescent proteins. A protein fused to Clontech’s Dendra2 glows green; activate it with a laser, and the color shifts to red. “Now you can distinguish the activated and nonactivated protein,” Haugwitz says.
With this capability, researchers can define a subset of proteins to follow, he explains. For example, you can aim the laser and activate red fluorescence in proteins in the endoplasmic reticulum. You can follow those proteins as they proceed to the Golgi body and then to other parts of the cell and beyond.
Labels don't need to be fancy to illuminate the cellular world, though. Even at their most basic, protein labels “opened up the molecular view of how the cell is put together,” Taraska says. “The Nobel Prize for green fluorescent protein was a big indication of how important this stuff has become.”
Compelling evidence, indeed.
Image: Clontech Laboratories