In situ proximity ligation assay for detection of proteins, their interactions and modifications

Abstract

To understand cellular processes and events responsible for their perturbations, proteomic analyses are needed in bio-medical research and clinical diagnostics. Several techniques based on specifically binding reagents (antibodies) or recombinant proteins (GFP fusion protein, methods of fluorescence/ bio-luminescence resonance energy transfer) are generally used to study protein location and activity resulting from secondary modifications and interactions. The in situ proximity ligation assay represents a novel technique of in situ protein imaging using DNA as a reporter molecule and DNA amplification processes. This method enables direct visualization of single molecules, their levels, modifications and pattern of interactions in individual fixed cells and tissues. Proximity probes consist of specific antibody with attached oligonucleotides that are used as reporter molecules for identification of such events. Proximity probes guide the formation of a circular DNA strand when bound in close proximity. The DNA circle after that serves as a template for rolling circle amplification allowing the interaction to be visualized. Compared to available proteomic techniques benefiting from genetic engineering, in situ PLA enables study of endogenous proteins in their natural environment and thus can be used for clinical specimens. The areas of applicability where proximity ligation procedure can be used include any research field where protein interaction measurements are important, such as signal-ing pathway studies, monitoring of pharmacological treatment targets and oncological diagnostics.