An (Immuno) Fluorescence Protocol for Monitoring Monoamine Oxidase A/B Protein Distribution Within the Cell.

ABSTRACT

The influence of a protein is not determined exclusively by its level of expression, but also by its localization within the cell. The literature often refers to the enzyme monoamine oxidase (MAO) as a mitochondrial enzyme, yet there is evidence that mitochondria-independent pools of MAO exist. These pools of MAO could exert distinct influences across physiological as well as pathological phenotypes. Fluorescence microscopy is a powerful tool for spatially resolving target proteins in cell and tissue preparations. This can rely on an antibody-based probe that targets the endogenous protein, e.g., immunofluorescence. In the event that antibodies might not be readily available or if one is interested in characterizing a variant of the wild-type protein, then a recombinant protein with a fluorescent fusion "tag" is preferred. We now describe a protocol for the detection of endogenous MAO using indirect immunofluorescence and a version of the protocol with minor modification for detecting (green) fluorescent protein-tagged MAOs. One observation we can highlight using these easily adaptable approaches is that MAO A and MAO B do not follow similar patterns of distribution throughout the cell, suggesting potential expression of MAO A and MAO B on distinct pools of mitochondria. Furthermore, distinct subcellular compartmentalization is suggested by the fact that a pool of MAO A, but not MAO B, is associated with certain lysosomal compartments. However, directed and quantitative studies will be required before any definitive statement can be made on these intriguing possibilities.