title: Use of BRET to Study Protein-Protein Interactions In Vitro and In Vivo.
abstract: Application of bioluminescence resonance energy transfer (BRET) assay has been of special value in measuring dynamic events such as protein-protein interactions (PPIs) in vitro or in vivo. It was only in the late 1990s the BRET assay using RLuc-YFP was introduced for biological research showing its use in determining interaction of two proteins involved in circadian rhythm. Several inherent attributes such as rapid and fairly sensitive ratiometric measurements, assessment of PPI irrespective of protein location in cellular compartment, and cost-effectiveness consenting to high-throughput assay development make BRET a popular genetic reporter-based assay for PPI studies. In BRET-based screening, within a defined proximity range of 10-100 Å, excited state energy of the luminescence molecule can excite the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using the Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP, or TurboFP as acceptor, have been reported for use.In recent years, BRET-related research has become significantly versatile in the assay format and its applicability by adopting the assay on multiple detection devices such as small-animal optical imaging platform or bioluminescence microscope. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular optical imaging applications based on BRET assays have broadened its scope for screening of pharmacological compounds by unifying in vitro, live cell, and in vivo animal/plant measurement all on one platform. Taking examples from the literature, this chapter contributes to in-depth methodological details on how to perform in vitro and in vivo BRET experiments, and illustrates its advantages as a single-format assay.
authors.affiliation: Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, India.
authors.forename: Shalini
authors.initials: S
authors.lastname: Dimri
authors.affiliation: Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, India.
authors.forename: Soumya
authors.initials: S
authors.lastname: Basu
authors.affiliation: Molecular Functional Imaging Lab, ACTREC, Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, India. ade@actrec.gov.in.
authors.forename: Abhijit
authors.initials: A
authors.lastname: De
mesh_terms: D000818:Animals; D001681:Biological Assay; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques; D049410:Luciferases, Renilla / Q000378:metabolism; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D061848:Optical Imaging / Q000379:methods*; D011485:Protein Binding; D060066:Protein Interaction Maps
chemical_list: D008164:Luminescent Proteins; D049410:Luciferases, Renilla
keywords: Bioluminescence resonance energy transfer; Cell-based assay; Fluorescent proteins; Luciferase; Optical imaging; Protein–protein interactions