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.
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:
- A pair of antibodies recognize and bind to two potentially interacting targets.
- These antibodies are conjugated to a matched pair (labeled +/-) of short single-stranded oligonucleotides.
- If the two respective targets interact, and hence remain in very close proximity, the oligonucleotide probes will hybridize and ligate with two additional “connector oligos” to form a continuous circular DNA structure.
- DNA polymerase enzymes will amplify these circular molecules through simple, reliable rolling-circle amplification.
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.
- Representative image of a proximity ligation assay.
- Performed with antibody pair ABIN1340332 against E2F2 & E2F1.
- Red fluorescence marks interaction between the two proteins. Blue fluorescence (DAPI) used as counterstain.
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 Simplifies Identification of Protein-Protein Interactions
- Simple - Proximity ligation assays don’t require weeks of molecular cloning or optimization to set-up and perform. They simply require availability of good probes and good antibodies.
- Sensitive - Readout in a proximity ligation assay is facilitated by detection of amplified DNA. The exponential nature of DNA amplification means that a very limited number of interacting molecules can produce a very strong, robust, visible signal. Sensitivity can be tuned by modulating amplification conditions (e.g. allowing additional time for rolling circle amplification to take place).
- In situ - Proximity ligation assays are performed directly in fixed cells and tissue sections. Researchers can characterize interaction in native protein, under near natural conditions. The proximity ligation assay does not require the researcher to destroy cells or extract protein content from tissue to identify protein-protein interactions, and other labels and tags can be used in the same to identify other variables simultaneously.
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.
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