Protein Localization with Streptavidin-Imaging: Boost Your Signal, Reduce Noise, Overcome Antibody Accessibility Problems, and Time-Travel to the Past.

Immunofluorescence experiments in Euglenozoa are more challenging than in cultured mammalian cells because they are smaller, often possess a protein-dense cellular envelope and antibodies are not commercially available. Here, we present streptavidin imaging as an alternative to immunofluorescence, that circumvents the above-mentioned problems, has several additional advantages and is applicable to all cells that can be genetically manipulated.For streptavidin imaging, the protein of interest is expressed fused to the biotin ligase TurboID and the (auto)biotinylation is detected with fluorescent streptavidin. In most cases, the streptavidin signal gives the same protein localization as an antibody but is superior to a classical immunofluorescence for three reasons. First, there is a significant enhancement of the signal due to multiple biotinylation sites, which is particularly useful when antigen levels are low, either due to low-level protein expression or as a result of procedures that reduce the antigen density as in expansion microscopy or correlative light and electron microscopy. Second, there is usually less unspecific signal in comparison to antibody labeling, as the streptavidin-biotin interaction is strong and highly specific. Third, streptavidin can access regions of the cell that are impermeable to most antibodies, in particular phase-separated areas like the nucleolus or the inner nuclear pore channel. In some cases, the streptavidin signal differs from the signal obtained by an antibody or a GFP fusion. The reasons are dynamic protein interactions with localization changes of either the bait protein or (some) interacting proteins. These differences between streptavidin and antibody signal can be exploited to understand protein dynamics and interactions in temporal and spatial resolution.Here, we present the protocol for streptavidin imaging: an all-in-one solution to antibody accessibility and low signal problems with the additional potential to study the dynamics of protein interactions.