Abstract
The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the available toolbox and ushered in a new era in biological electron microscopy. Here, we describe methods for combining conditionally-stable nanobodies to fluorescent protein tags with APEX-based detection. These methods are compatible with detection of low levels of expression of fluorescently-tagged proteins and with detection of protein complexes using split GFP-based complementation methods. We describe a simple protocol for applying these methods to the electron microscopic detection of proteins and protein complexes in cultured cells.
Keywords: Electron microscopy, GFP, Nanobodies, APEX, Peroxidase, Localization
Background
Recently, we developed a system for the detection of GFP-labeled proteins in transfected cells by electron microscopy (Ariotti et al., 2015). This simple system involves the co-transfection of a plasmid encoding a GFP binding nanobody; a small, genetically encodable, single chain antibody (Kubala et al., 2010), fused to an ascorbate peroxidase APEX2-tag (Martell et al., 2012), which is able to catalyze a reaction to produce a precipitate visible in the electron microscope. More recently, we have expanded the scope of this methodology with i) the addition of an mCherry-binding nanobody allowing detection of mCherry-tagged proteins, ii) the adoption of conditionally stable nanobody sequences and iii) the application to bimolecular fluorescence complementation (Hu et al., 2002), allowing detection of pairwise interactions at the ultrastructural level (Ariotti et al., 2018).
Materials and Reagents
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Acknowledgments
This work was supported by grants and a fellowship from the National Health and Medical Research Council of Australia (grant numbers APP1037320, APP1058565, and APP569542 to RGP; APP1045092 to RGP and NA; APP1099251 to TEH and RGP). The use of APEX for EM is based on the development and application of APEX2 as described by Lam et al. (2015). Destabilized nanobodies are based on the studies of Tang et al. (2016).
Competing interests
The authors declare that they have no conflicts of interest and no competing interests.
References
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