This method enables high-resolution cell imaging, using either light or electron microscopy, and is especially useful for probing protein-protein interactions.
Determining protein localization in live cells while maintaining sample integrity is difficult. Traditional techniques involve antibody staining of target proteins, but the permeablization treatments required for antibody access compromise cellular ultrastructure. Alternatives are limited by an inconvenient requirement for light and oxygen gas (which restricts their use to thin samples), or lack of activity in most cellular compartments.
Professor Alice Ting and colleagues have developed a tool that involves genetically fusing an engineered ascorbate peroxidase (APEX), to a protein of interest to catalyze the production of an electron-dense precipitate that is easily visualized with either light or electron microscopy. This reaction occurs quickly and locally, thereby attaining very high spatial resolution. The tag is non-toxic, is functional in all cellular compartments, and does not sterically hinder native proteins. Furthermore, it can be fused to fluorescent proteins, allowing for images from electron microscopy to be correlated with those from light microscopy. APEX2, an improved version of this enzyme, will be available shortly.
High spatiotemporal resolution
Can be used to combine multiple imaging modalities
Genetic tag is small, sterically compatible with most proteins, and can be expressed in all cellular compartments
Can be used on samples of any thickness (reaction is not light-dependent)