Jul 22, 2019 | Updated: 09:15 AM EDT

A Unique Tool for Live-Cell Imaging

Jul 03, 2019 09:13 PM EDT

A Unique Tool for Live-Cell Imaging
(Photo : Ning Zhao/Colorado State University)

The biomolecules human immune systems deploy to find, tag, and destroy invading pathogens are the antibodies. Their work includes binding to specific targets, called epitopes, on the surfaces of antigens, like locks to keys. Scientists have, for so many years, 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.

Epitope tagging is one tried and true technique that 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.

Recently, a cross-disciplinary group of researchers from Colorado State University and the Tokyo Institute of Technology has made an addition to the new tool in the arsenal of antibody-based probes, but with a powerful distinction: the researchers' genetically encoded probe works in living cells. Professor Tim Stasevich from CSU Monfort led the work and also Professor Hiroshi Kimura from Tokyo Tech, and they published the result of their study in the journal Nature Communications.

The first author of the study and a post-doctoral researcher in Stasevich's lab who designed most of the experiments, Ning Zhao, noted that their new antibody-based probe is affectionately called a "frankenbody." It is 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.

An assistant professor in the Department of Biochemistry and Molecular Biology at CSU, Stasevich, said that they are interested in intracellular antibodies because they can use them as imaging reagents in a live cell. Tagging is not necessary, as a Green Fluorescent Protein, instead there is a fluorescent antibody that will bind to the protein that needs to be visualized.

The new probe would be a useful complement to the green fluorescent protein (GFP), a comprehensive biochemistry tool and subject of a Nobel Prize that involves genetically fusing a light-up green tag to a protein of interest. The GFP, however, is limited by its relatively large size and the time it takes to fluoresce. With this new probe from the CSU researchers, the tag is smaller and becomes fluorescent faster, so the "birth" of a protein of interest can be captured in real time.

The scientists have the goal of making their tool useful immediately, and they designed their probe to work with the classic HA tag. HA is a widely used small linear epitope tag that is derived from a portion of the human influenza virus protein hemagglutinin. According to Stasevich, for the longest time, people have been looking at HA-tagged proteins in fixed, dead cells. Now, it is possible to image the dynamics of those proteins in live cells.

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