Many infectious viruses, from HIV to West Nile, rely on a fundamental biological process called frameshifting to maximize their attack. Long identified as a key mechanism that viruses use to proliferate inside their hosts, the real-time dynamics of frameshifting had never been directly obse ... more
It's not an antibody, it's a frankenbody: A new tool for live-cell imaging
Antibody-based probe works in living systems and targets the classic HA tag
Antibodies are the biomolecules our immune systems deploy to find, tag and destroy invading pathogens. They work by binding to specific targets, called epitopes, on the surfaces of antigens - like locks to keys.
For many decades, scientists have cleverly exploited this selective tagging mechanism in natural antibodies to engineer antibody-based probes that let them purify and study different types of proteins within cells. One tried and true technique, epitope tagging, involves fusing an epitope to a protein of interest and using fluorescently labeled antibodies to make those proteins visible - but only in fixed, dead cells.
Now, a cross-disciplinary team of researchers from Colorado State University and the Tokyo Institute of Technology have added a new tool to the arsenal of antibody-based probes, but with a powerful distinction: Their genetically encoded probe works in living cells. The work, led by CSU Monfort Professor Tim Stasevich and Tokyo Tech Professor Hiroshi Kimura, is described in Nature Communications.
According to first author Ning Zhao, a postdoctoral researcher in Stasevich's lab who designed most of the experiments, their new antibody-based probe is affectionately called a "frankenbody." Like stitching new limbs on a body, the scientists have taken the binding regions of a normal antibody, the "sticky parts," and grafted them to a different scaffold that remains stable in live cells but retains the specificity of the antibody.
"We're interested in intracellular antibodies because you can use them as imaging reagents in a live cell," said Stasevich, an assistant professor in the Department of Biochemistry and Molecular Biology at CSU. "You don't need a tag, like a Green Fluorescent Protein, because instead you have this fluorescent antibody that will bind to your protein that you want to visualize."
The new probe would be a useful complement to the green fluorescent protein (GFP), a widespread biochemistry tool and subject of a Nobel Prize that involves genetically fusing a light-up green tag to a protein of interest. However, the GFP is limited by its relatively large size and the time it takes to fluoresce; with the CSU researchers' new probe, the tag is smaller and becomes fluorescent faster, so the "birth" of a protein of interest can be captured in real time.
With the goal of making their tool immediately useful, the scientists designed their probe to work with the classic HA tag. HA is a widely used small linear epitope tag that's derived from a portion of the human influenza virus protein hemagglutinin.
"For the longest time, people have been looking at HA-tagged proteins in fixed, dead cells," Stasevich said. "Now we can image the dynamics of those proteins in live cells."
The possibilities of how scientists may use the new probe are limitless. Stasevich's lab is particularly interested in studying RNA translation, and they plan to use their new system to more easily design new RNA imaging experiments.
Added Zhao, the HA tag is tiny - a chain of just nine amino acids - and the probe is genetically encoded on a plasmid that can be easily transferred into a cell. This is in contrast to traditional antibodies, which can cost a lab several hundreds of dollars per order, suffer from lot-to-lot variability, and are difficult to get into cells. The new probe from Stasevich's team therefore provides a low-cost solution for protein and RNA translation imaging.
In the paper, the scientists demonstrated some applications, including single-protein tracking, single-RNA translation imaging, and amplified fluorescence imaging in zebrafish embryos. All of these experiments are more challenging when using traditional fluorescent protein tags.
"We have several new imaging reagents in the works that build off of this success, so I see great things ahead," Stasevich said.
- live cell imaging
On the surfaces of our trillions of cells is a complex crowd of molecules moving around, talking to each other, occasionally segregating themselves, and triggering basic functions ranging from pain sensation to insulin release. The structures that organize this microscopic traffic jam are n ... more
The world's most advanced light microscopes allow us to see single molecules, proteins, viruses and other very small biological structures. But even the best microscopes have their limits. Colorado State University scientists are pushing the limits of a technique called super-resolution mic ... more
Scientists at Tokyo Institute of Technology (Tokyo Tech) developed a new methodology that allows researchers to assess the chemical composition and structure of metallic particles with a diameter of only 0.5 to 2 nm. This breakthrough in analytical techniques will enable the development and ... more
Scientists have successfully visualized terahertz radiation, popularly known as T-rays, using a crystal called mayenite (Ca12Al14O33). Their method cleverly utilizes the rattling motion caused by the vibration of oxygen ions inside the cage-like structures of the crystal. In recent years, t ... more
Each odor-detecting neuron (referred to as olfactory sensory neuron from here on), chooses a single odorant receptor gene from a fairly large number of options that are split into class I (fish-like) and class II (terrestrial-specific) odorant receptors. This strict selectiveness of sensory ... more
- 1Virtual screening for active substances against the coronavirus
- 2analytica 2020 is postponed
- 3FDA Provides Emergency Use Authorization to PerkinElmer for COVID-19 Testing
- 4Roche’s cobas SARS-CoV-2 Test to detect novel coronavirus receives FDA Emergency Use Authorization
- 5The digital laboratory live and tangible
- 6Smartphone lab finds coronavirus in saliva
- 7Thermo Fisher Scientific to Acquire QIAGEN
- 8Pool testing of SARS-CoV-02 samples increases worldwide test capacities many times over
- 9Abbott Receives FDA Emergency Use Authorization and Launches Test to Detect Novel Coronavirus
- 10Bosch develops rapid test for COVID-19
- New blood test detects wide range of cancers
- Staining Cycles with Black Holes
- FDA Provides Emergency Use Authorization to PerkinElmer for COVID-19 Testing
- New device quickly detects harmful bacteria in blood
- Abbott Receives FDA Emergency Use Authorization and Launches Test to Detect Novel Coronavirus