Fast Screening of Protein-Protein Interactions Using Forster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells

Durhan, Seyda Tugce
Sezer, Enise Nalli
Son, Çağdaş Devrim
Küçük Baloğlu, Fatma
Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Forster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.


Balkan, Seyda Tuğçe; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biochemistry (2021-8-11)
GPCR’s are seven-transmembrane receptors that transmit external signals to the intracellular environment via secondary messenger systems through heterotrimeric G proteins. Heterotrimeric G proteins consist of α and β-γ subunits. Until recent years, scientists thought that GPCR signal transduction occurs between one GPCR and one heterotrimeric G protein; however, recently, it has been shown that GPCR’s can make oligomers. Oligomerization of GPCR allows cells to tune the intensity of the signal and respond ap...
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Tasel, Serdar F.; HASSANPOUR, REZA; Mumcuoğlu, Ünal Erkan; Perkins, Guy; Martone, Maryann (2014-02-18)
Mitochondria are sub-cellular components which are mainly responsible for synthesis of adenosine tri-phosphate (ATP) and involved in the regulation of several cellular activities such as apoptosis. The relation between some common diseases of aging and morphological structure of mitochondria is gaining strength by an increasing number of studies. Electron microscope tomography (EMT) provides high-resolution images of the 3D structure and internal arrangement of mitochondria. Studies that aim to reveal the c...
Parallelization of functional flow to predict protein functions
Akkoyun, Emrah; Can, Tolga; Department of Medical Informatics (2011)
Protein-protein interaction networks provide important information about what the biological function of proteins whose roles are unknown might be in a cell. These interaction networks were analyzed by a variety of approaches by running them on a single computer and the roles of the proteins identified were used to predict the function of the proteins unidentified. The functional flow is an approach that takes the network connectivity, distance effect, topology of the network with local and global views int...
Investigation of Gai1 protein homodimerization in live cells using förster resonance energy transfer (FRET) and biomolecular fluorescence complementation assay (BIFC)
Atay, Özge; Son, Çağdaş Devrim; Department of Biochemistry (2019)
The classical GPCR signaling pathway, where a heterotrimeric G protein-GPCR interaction is sufficient to transmit the signal to effector proteins has been replaced by a heteromeric G protein-GPCR homo- or hetero-dimer interaction model over the past two decades. These studies demonstrate that GPCRs that interact with each other couple with a heteromeric G protein. In recent years, evidence suggests that dimer of GPCR dimers is required for some complex signal transductions. In these studies, it was proposed...
Visualization of interactions between fluorescently tagged g protein α11, α12/13 subtypes and adenosine 2a, dopamine 2 or homodimer adenosine 2a/2a receptors
Kostromin, İrmak Begüm; Son, Çağdaş Devrim; Department of Biology (2018)
G-Protein-coupled receptors (GPCRs) belong to one of the largest family of cell surface receptors, which transmit extracellular signals to intracellular responses by interacting with G- proteins. The G proteins are known as molecular switches that regulates different pathways via control of secondary messengers and signaling proteins. Adenosine 2A (A2A) and Dopamine 2 (D2) receptors belong to G-Protein-coupled receptors (GPCRs) family and are located mostly in striatopallidal γ-aminobutyric acid (GABA) cont...
Citation Formats
S. T. Durhan, E. N. Sezer, Ç. D. Son, and F. Küçük Baloğlu, “Fast Screening of Protein-Protein Interactions Using Forster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells,” APPLIED SPECTROSCOPY, pp. 0–0, 2022, Accessed: 00, 2023. [Online]. Available: