Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA)

Maria Magdalena Mocanu, Tímea Váradi, János Szöllosi, Peter Nagy

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2063-2070
Number of pages8
JournalProteomics
Volume11
Issue number10
DOIs
Publication statusPublished - May 1 2011

Keywords

  • Cell biology
  • Flow cytometry
  • Fluorescence resonance energy transfer
  • Protein associations
  • Proximity ligation assay

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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