Figure 1. Schematic presenting the principal of the Proximity Ligation Assay® (PLA) for the detection of two proteins within a proximity of ≤ 40nm.

(1) Two antibodies of different species are selected that recognize two proteins of interest. (2) Secondary antibodies that recognize the Fc regions of the primary antibody species are conjugated with oligonucleotide probes and then incubated with the sample. (3) Ligase and free oligonucleotides that hybridize with the probes bring the oligonucleotides together in a closed circle confirmation if the proteins are in close enough proximity. (4) Finally, polymerase is added to the reaction to allow for rolling circle amplification, while fluorescently labeled oligonucleotides hybridize with the rolling circle product resulting in the accumulation of a fluorescent spot in areas of successful amplification.