title: Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA).
abstract: Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.
authors.affiliation: Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary.
authors.forename: Maria-Magdalena
authors.initials: MM
authors.lastname: Mocanu
authors.forename: Tímea
authors.initials: T
authors.lastname: Váradi
authors.forename: János
authors.initials: J
authors.lastname: Szöllosi
authors.forename: Peter
authors.initials: P
authors.lastname: Nagy
mesh_terms: D000345:Affinity Labels / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D061067:Antibodies, Monoclonal, Humanized; D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D016014:Linear Models; D017711:Nonlinear Dynamics; D025941:Protein Interaction Mapping / Q000379:methods*; D000068878:Trastuzumab
chemical_list: D000345:Affinity Labels; D000906:Antibodies; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D002232:Carbocyanines; C085321:cyanine dye 5; D000068878:Trastuzumab