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PLoS Biol. 2018 Apr 5;16(4):e2005473. doi: 10.1371/journal.pbio.2005473. eCollection 2018 Apr.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

Author information

1
The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia.
2
EMBL Australia Node in Single Molecule Sciences, School of Medical Science, The University of New South Wales, Sydney, New South Wales, Australia.
3
The University of Queensland, Centre for Microscopy and Microanalysis, Brisbane, Queensland, Australia.

Abstract

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

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