Live Cell Labeling Background

Labeling of proteins inside living cells is highly useful in the characterization of protein dynamics, mobility and function and ultimately in the understanding of biological processes. Labelling proteins with fluorescent proteins such as GFP to monitor their cellular localization has been described since the mid-1990’s. However, expression of proteins fused to GFP has significant drawbacks as these fusion proteins can have altered localization and altered function, due to their increased size. Chemical labelling of proteins is more versatile than labelling via fusion proteins and opens the possibility of labelling with a wide array of functionalities, most notably fluorescence, with bright organic dyes of a broad range of colours (Schneider & Hackenberger, 2017). Chemical labels can also be used in conjunction with bioorthogonal labeling methods to image and retrieve nucleic acids, proteins, glycans, lipids and other metabolites in vitro, in cells as well as in whole organisms.

Metabolic chemical reporters are non-natural chemical compounds which can be introduced into naturally occurring biomolecules of a living system, usually through the cell’s biosynthetic machinery. These chemical reporters are then reacted with specifically designed molecular probes in bioorthogonal labeling reactions that allow the visualization and/or isolation of biomolecules of interest. However, the chemical reporter groups have to be stable before the reaction occurs and nontoxic to the organism. Moreover, the reaction between the chemical reporter groups and the probes should occur selectively under physiological conditions and both should be inert to the myriad of chemical reactions which naturally occur in cells (Kim, 2018).

One example, is metabolic glycan labeling which can be used for imaging glycans in living organisms. This strategy relies on the cellular biosynthetic machinery incorporating a modified monosaccharide that contains a bioorthogonal chemical reporter. The metabolic incorporation of this reporter into glycans can be further visualized by chemical ligation (click chemistry) with a label, such as a fluorescent probe. Labelled cells can then be visualised using flurescence microscopy/spectroscopy, confocal microscopy, flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) etc. Cells can also be coupled to magnetic beads for pulldown experiments or other tags, such as biotin, for isolation and purification. Click chemistry armed ELISAs have also been described (Lanyon-Hogg et al., 2015).

References

• Lanyon-Hogg et al. (2015). Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase. Analytical biochemistry, 490, 66–72.
• Schneider, A. F. L., & Hackenberger, C. P. R. (2017). Fluorescent labelling in living cells. Current Opinion in Biotechnology, 48, 61–68.
• Kim, E.J. (2018). Chemical Reporters and Their Bioorthogonal Reactions for Labeling Protein O-GlcNAcylation. Molecules, 23(10), 2411.