ABSTRACT
Immunostaining has recently been used to map expression of all proteins in various types of tissues and diseases. However, obtaining information on functional states of proteins, such as protein complexes and posttranslational modifications (PTMs), calls for other approaches. Methods that do allow spatial identification of protein-protein interactions are required to determine the signalling network profile of each cell, as this provides the foundation for analysis of cellular communication. Forster resonance energy transfer (FRET) can be used for such analysis. Nonetheless, although antibody-based FRET can be used to analyze protein-protein interactions, the method is mainly used by expressing pairs of fluorescent fusion proteins, which requires genetic manipulation of the cells. Alternatively In situ proximity ligation assay can be used for highly specific detection of single proteins, protein-protein interactions, and PTMs such as phosphorylation that is informative for determining the activation state of signalling cascades within the cell.
