Caught on Camera with Another Protein – Just Good Friends or Something More? A Guide to Localization, Colocalizaton and Interaction on the Light Microscope

become more advanced yet are increasingly marketed as being simple to use. In the same time period, there has also been a general shift in microscopy provision in academic institutes from a “single lab, single microscope” model to a centralised approach, resulting in the onus on training and quality control often falling on facility staff. Here we attempt to provide an example of how the interaction between facility and research staff can lead to improvements in image quality and reduce the likelihood of a member of research staff “over interpreting” their data. In this case, we have described a work flow from the fairly simple job of identifying the localization of a single protein within the cell, through the practicalities of doing a colocalization experiment for 2 fluorescently tagged proteins, ending with the use of a fluorescence resonance energy transfer (FRET) experiment to determine whether these colocalized proteins are actually interacting.