Improved HaloTag Ligand Enables BRET Imaging With NanoLuc.
BRET imaging
HaloTag
Janelia Fluor dyes
NanoLuc
PKA
Journal
Frontiers in chemistry
ISSN: 2296-2646
Titre abrégé: Front Chem
Pays: Switzerland
ID NLM: 101627988
Informations de publication
Date de publication:
2019
2019
Historique:
received:
14
10
2019
accepted:
23
12
2019
entrez:
30
1
2020
pubmed:
30
1
2020
medline:
30
1
2020
Statut:
epublish
Résumé
Bioluminescence resonance energy transfer (BRET) from an exceptionally bright luciferase, NanoLuc, to a fluorescent HaloTag ligand is gaining momentum to monitor molecular interactions. The recommended use of HaloTag618 ligand for the NanoLuc-HaloTag BRET pair is versatile for ensemble experiments due to their well-separated emission bands. However, this system is not applicable for single-cell BRET imaging because of its low BRET efficiency and in turn weak acceptor signals. Here we explored the unprecedented potential of rhodamine based HaloTag ligands, containing azetidine rings, as BRET acceptors. Through a comprehensive evaluation of various commercial and Janelia Fluor HaloTag ligands for improved BRET efficiency and minimal donor signal bleed-through, we identified JF525 to be the best acceptor for microscopic BRET imaging. We successfully employed BRET imaging with JF525 to monitor the interaction of protein kinase A catalytic and regulatory subunit. Single-cell BRET imaging with HaloTag JF525 can henceforth open doors to comprehend and interpret molecular interactions.
Identifiants
pubmed: 31993413
doi: 10.3389/fchem.2019.00938
pmc: PMC6970966
doi:
Types de publication
Journal Article
Langues
eng
Pagination
938Informations de copyright
Copyright © 2020 Thirukkumaran, Wang, Asouzu, Fron, Rocha, Hofkens, Lavis and Mizuno.
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