Detection of protein interactions by Subcellular Localization Assay

SEVERAL techniques have been developed to study protein association or interactions, such as co-immunoprecipitation, co-localization, fluorescence resonance transfer energy (FRET) or yeast-two-hybrid system (1). Nevertheless, each of the aforementioned techniques has its advantages and disadvantages. Patient-derived tissue samples are unique, in a sense they cannot be modified by exogenous expression or overexpression of genetically modified proteins and they typically have high background fluorescence intensity (1,2). To circumvent all these impediments, a Swedish research group developed Proximity Ligation Assay (PLA), a proteomic tool which allows the visualization of protein interactions and their posttranslational modifications with high sensitivity with the help of amplified DNA (3,4). The technique was initially established as a tool to detect cytokines (5,6) and was further adapted to study protein post-translational modification in situ (2,3,7). The PLA method uses two antibodies conjugated with oligonucleotides (proximity probes), each antibody being specific for a protein of interest or a cellular structure (3). Two connector oligonucleotides are added to the reaction mixture leading to the formation of circular DNA followed by joining the free ends of DNA by the addition of DNA ligase. In the next step, Rolling Circle Amplification (RCA) will generate a single-stranded DNA product which is connected to one of the proximity probes and it contains several concatenated copies of the connector oligonucleotides. Detection of the amplified DNA is carried out by hybridization with a fluorescently conjugated oligonucleotide (detection oligonucleotides) complementary to a short segment of the connector oligonucleotide appearing as a bright spot having a high signal-tonoise ratio in fluorescent microscopy (2,3,6). Interpretation of the results of PLA are based on the assumption that a PLA signal is detectable if the proximity probes are located at a distance of tens of nanometers (7,8). However, PLA was also considered a “molecular ruler” suitable for measuring distances between two epitopes in the range of 10–30 nm (3). Nevertheless, for the quantitative measurements in the range of 2– 10 nm, F€ orster resonance energy transfer (FRET) should be considered as a more reliable tool (9–13) and at the same time, the saturation features of PLA should be taken into account (14). It is known that photobleaching or autofluorescence of tissue samples can represent drawbacks for FRET, so researchers should take these facts into account when selecting the optimal method to answer their scientific questions (1). Nevertheless, a couple of measurement techniques can minimize the autofluorescence effects on FRET. The accuracy of FRET measurements can be improved by cell-by-cell correction for autofluorescence (15) and by the use of FRET in combination with another technique: total internal reflection fluorescence microscopy (16). The development of new instruments, faster computers and different software tools led to the adaptation of PLA to a statistically more robust technique, flow cytometry (2,8).