Do molecules A and B interact?
This is a profoundly simple question that, in certain instances, can present a significant challenge to the cell-biologist.
Traditional methods for identifying protein-protein interactions include a host of complex experiments like two-hybrid assays and co-immunoprecipitations. While these experiments do provide the opportunity to assay interaction, they are not without several well-known and inherent limitations.
For example, two-hybrid assays commonly require exogenous expression of target fusion-proteins and identify interaction only under artificial conditions. By contrast, co-immunoprecipitation, performed in vitro, can be used to identify interaction between endogenously expressed native proteins (availability of antibodies notwithstanding), but necessitates a moderate to high concentration of both target proteins in order to generate effective readout.
The proximity ligation assay (PLA) is another sensitive, simple, in situ tool in the researcher’s toolbox that can help provide insight into protein-protein interactions.
What is a proximity ligation assay?
A proximity ligation assay is an in situ marriage between traditional antibody-based affinity proteomics techniques and modern molecular biology.
In a proximity ligation assay:
The result is a highly amplified circular DNA molecule that can be detected via standard fluorescent methods, and that acts as a qualitative marker of interaction between the two proteins.
Why use a PLA?
No single technique for identifying protein-protein interactions offers an absolute gold-standard, and each method has its own strengths and limitations. Researchers who study protein-protein interaction will often perform several different competing experiments in order to show the same interaction through multiple channels.
The PLA is an attractive proposition for identifying protein-protein interactions because it is:
The proximity ligation assay is a simple, qualitative method for identifying protein-protein interactions in situ. The assay has also been co-opted to identify post translational modifications like phosphorylation.
Leveraged properly, with a firm understanding of the assay strengths and limitations, the PLA provides yet another powerful tool for the researcher who wishes to foment a more complete understanding of intracellular dynamics and protein interaction.