26 ANALYTICAL AND LABORATORY EQUIPMENT
Determining
PROTEIN INTERACTIONS M
any molecular
Tobias Pusterla PhD reports on monitoring protein- protein interactions in high-throughput formats
biologists focus their research on cellular
responses to external stimuli. Receptors, such as G protein- coupled receptors (GPCRs), act as mediators between the extracellular environment and the intracellular response.
accumulates in the cell upon ligand-receptor binding (Fig. 1). An established method to determine protein-protein interactions is Bioluminescence Resonance Energy Transfer (BRET). In BRET, luciferase is coupled to one protein and a fluorophore to another. Protein interaction is detected upon addition of the luciferase substrate. If a protein-protein interaction occurs, the luciferase emission light excites the fluorophore of the bound protein, which emits at a different wavelength, the BRET signal.
An international research
Fig. 1. GPCR activation results in accumulation of second messengers cAMP or IP1
REFERENCES: 1
Lundstrom K. (2009) An
overview on GPCRs and drug discovery: structure- based drug design and strutural biology on GPCRs. Methods Mol Biol. 552:51-66. 2
Stoddart et al. (2015)
Application of BRET to monitor ligand binding to GPCRs. Nat. Methods 12(7): 661-663.
www.scientistlive.com
Te importance of GPCRs is highlighted by the fact that they are targeted by 30-50% of all commercially available pharmaceuticals.1 Traditionally, radioactively labelled ligands were used to detect protein-ligand interactions as they provide very precise results. However, the primary reason for looking for alternative methods is the fact that radioactive measurements are not compatible with high- throughput formats.
Ligand-binding measurements save time Non-radioactive techniques such as fluorescence and luminescence are ideal for high-throughput measurements in microplates. Researchers can either directly measure the ligand-receptor interaction or quantify the concentration of a second messenger, which temporarily
group recently developed a BRET-based technology that extends researchers’ capability to allow assessment of real-time binding kinetics in living cells.2 Te project team consists of the laboratories of associate professor Kevin Pfleger (University of Western Australia/Harry Perkins Institute of Medical Research), professor Stephen Hill (University of Nottingham), Promega with its latest reagents, and BMG Labtech providing its leading high-throughput multi-mode microplate readers, Clariostar and Pherastar FS. Nano
luciferase (NanoLuc) was expressed at the
N-terminus of a GPCR while the ligands were labelled with a fluorophore. As BRET signal occurs when the ligand binds to the receptor, the measurements enable the monitoring of a specific ligand binding to a variety of GPCRs (e.g. adreno or angiotensin receptor). In contrast, cells transfected
with another construct, the well-known Renilla Luciferase (RLuc8), didn’t show specific binding. Tis is credited to the fact that the considerably smaller NanoLuc can be effectively transported to the cell membrane, whereas Renilla constructs cannot because of their size.
Requirements for detection systems Automation and miniaturisation are key features in drug discovery. BMG Labtech’s microplate readers are equipped with integrated injectors to add substances during the measurement, simultaneous dual emission detection, and can be easily integrated into robotic systems. Moreover, the dedicated analysis software (MARS) that automatically calculates and displays assays parameters like IC50
or S/B values helps to
decrease data analysis time. For more information ✔ at
www.scientistlive.com/eurolab
Fig. 2. Multi-mode microplate readers Clariostar and Pherastar FS
Tobias Pusterla PhD is with BMG Labtech.
www.bmglabtech.com
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