This chapter discusses recent advances regarding biomolecular interaction detection strategies based on bioluminescence. The commonly used transcriptional approaches, such as yeast two hybrid assays and ubiquitin split, based on transcriptional activation of reporter genes following target protein interaction in the nucleus, have been replaced by more suitable strategies that allow to monitor interactions occurring in every cell compartment. Many bioanalytical tools for in vivo and in vitro applications have been proposed, mostly based on resonance energy transfer (RET) process, such as Fluorescence and Bioluminescence Resonance Energy Transfer (FRET and BRET). The applicability of BRET, thanks to its advantages with respect to the analogous FRET, to elucidate protein dynamics in living cells has been widely explored using different donors and acceptors, including quantum dots. In addition, the split luciferase complementation approach has been successfully employed for monitoring protein interactions in vivo and showed able to detect even weak interactions. Also combinations of these approaches, such as sequential BRET-FRET or split complementation-FRET, have been explored to detect more than two interacting proteins, but their analytical performance still need improvements and surely new ways of monitoring protein interactions will certainly emerge.

Biomolecular interactions

MICHELINI, ELISA;CEVENINI, LUCA;MEZZANOTTE, LAURA;RODA, ALDO
2010

Abstract

This chapter discusses recent advances regarding biomolecular interaction detection strategies based on bioluminescence. The commonly used transcriptional approaches, such as yeast two hybrid assays and ubiquitin split, based on transcriptional activation of reporter genes following target protein interaction in the nucleus, have been replaced by more suitable strategies that allow to monitor interactions occurring in every cell compartment. Many bioanalytical tools for in vivo and in vitro applications have been proposed, mostly based on resonance energy transfer (RET) process, such as Fluorescence and Bioluminescence Resonance Energy Transfer (FRET and BRET). The applicability of BRET, thanks to its advantages with respect to the analogous FRET, to elucidate protein dynamics in living cells has been widely explored using different donors and acceptors, including quantum dots. In addition, the split luciferase complementation approach has been successfully employed for monitoring protein interactions in vivo and showed able to detect even weak interactions. Also combinations of these approaches, such as sequential BRET-FRET or split complementation-FRET, have been explored to detect more than two interacting proteins, but their analytical performance still need improvements and surely new ways of monitoring protein interactions will certainly emerge.
Chemiluminescence and bioluminescence: past, present and future
378
397
E. Michelini; L. Cevenini; L. Mezzanotte; A. Coppa; A. Roda
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/99980
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