Proximity Ligation Assay (PLA) to Detect Protein-protein Interactions in Breast Cancer Cells

Abstract

Protein-protein interaction networks provide a global picture of cellular function and biological processes, and the dysfunction of some interactions causes many diseases, including cancer. The in situ proximity ligation assay (PLA) is a powerful technology capable of detecting the interactions among proteins in fixed tissue and cell samples. The interaction between two proteins is detected using the corresponding two primary antibodies raised in different species. Species-specific secondary antibodies (PLA probes), each with a unique short DNA strand attached to it, bind to the primary antibodies. When the PLA probes are in close proximity (<40 nm), the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides. Several-hundredfold replication of the DNA circle can occur after the amplification reaction, and a fluorescent signal is generated by labelled complementary oligonucleotide probes. Therefore, each detected signal is visualized as an individual fluorescent dot, which can be quantified and assigned to a specific subcellular location based on microscopy images. This revolutionary technique enables us to study the protein complex formation with high specificity and sensitivity compared to the other traditional methods, such as co-immunoprecipitation (Co-IP).