期刊
ANALYSIS & SENSING
卷 2, 期 6, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/anse.202200020
关键词
fluorescent proteins; FRET; immunoassays; luminescence; nanobodies
资金
- NSF [1830941]
- National Science Foundation Graduate Research Fellowship [DGE1745303]
The article presents a new strategy based on CoraFluor-1-functionalized nanobodies to simplify the installation of TR-FRET donor fluorophores on target proteins. This strategy is important for the design and development of TR-FRET assays.
Owing to their exceptional sensitivity and specificity, assays based on time-resolved Forster resonance energy transfer (TR-FRET) have become increasingly popular in biomedical research. TR-FRET assays are highly versatile and compatible with complex biological environments. However, the paucity of straightforward strategies for the selective installation of TR-FRET donor fluorophores on target proteins, which is a critical requirement for most assay designs, has limited the potential and adaptation of this powerful technology. To address this issue, we have developed a versatile toolbox strategy based on CoraFluor-1-functionalized nanobodies. The target-agnostic modular design simplifies the TR-FRET donor-labeling of epitope tags, fluorescent proteins and primary antibodies, including their use with endogenous proteins in lysates. Our strategy, which we extend to enable the seamless adaptation of split-luciferase systems for TR-FRET approaches, greatly facilitates the development and design of TR-FRET assays for both target engagement studies and the quantification of proteins of interest with sub-nanomolar sensitivity.
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