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Protocol for deriving proximity, affinity, and stoichiometry of protein interactions using image-based quantitative two-hybrid FRET.
- Source :
-
STAR protocols [STAR Protoc] 2023 Sep 15; Vol. 4 (3), pp. 102459. Date of Electronic Publication: 2023 Jul 28. - Publication Year :
- 2023
-
Abstract
- Two-hybrid Förster resonance energy transfer (FRET) provides proximity, affinity, and stoichiometry information in binding interactions. We present an image-based approach that surpasses traditional two-hybrid FRET assays in precision and robustness. We outline instrument setup and image acquisition and further describe steps for image preprocessing and two-hybrid FRET analysis using provided software to simplify the workflow. This protocol is compatible with confocal microscopes for high-precision and imaging plate readers for high-throughput applications. A plasmid-based reference system supports fast establishment of the protocol. For complete details on the use and execution of this protocol, please refer to Rivas et al. <superscript>1</superscript> .<br />Competing Interests: Declaration of interests The authors declare no competing interests.<br /> (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Plasmids
Fluorescence Resonance Energy Transfer methods
Software
Subjects
Details
- Language :
- English
- ISSN :
- 2666-1667
- Volume :
- 4
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- STAR protocols
- Publication Type :
- Academic Journal
- Accession number :
- 37516972
- Full Text :
- https://doi.org/10.1016/j.xpro.2023.102459