PUBLICATION
Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors
- Authors
- Kardash, E., Bandemer, J., and Raz, E.
- ID
- ZDB-PUB-111117-13
- Date
- 2011
- Source
- Nature Protocols 6(12): 1835-1846 (Journal)
- Registered Authors
- Bandemer, Jan, Kardash, Elena, Raz, Erez
- Keywords
- cell biology, developmental biology, imaging, model organisms
- MeSH Terms
-
- Algorithms
- Animals
- Biosensing Techniques/methods*
- Embryo, Nonmammalian/chemistry
- Embryo, Nonmammalian/cytology
- Embryo, Nonmammalian/metabolism*
- Fluorescence Resonance Energy Transfer/methods*
- Proteins/analysis*
- Proteins/genetics
- Proteins/metabolism
- RNA, Messenger/analysis
- Zebrafish/embryology
- Zebrafish/genetics
- Zebrafish/metabolism
- PubMed
- 22051797 Full text @ Nat. Protoc.
Citation
Kardash, E., Bandemer, J., and Raz, E. (2011) Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nature Protocols. 6(12):1835-1846.
Abstract
Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2–3 d.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping