Abstract:
:This protocol describes procedures for performing fluorescence resonance energy transfer (FRET) microscopy analysis by three different methods: acceptor photobleaching, sensitized emission and spectral imaging. We also discuss anisotropy and fluorescence lifetime imaging microscopy-based FRET techniques. By using the specific example of the FRET probe Akind (Akt indicator), which is a version of Akt modified such that FRET occurs when the probe is activated by phosphorylation, indicating Akt activation. The protocol provides a detailed step-by-step description of sample preparation, image acquisition and analysis, including control samples, image corrections and the generation of quantitative FRET/CFP ratio images for both sensitized emission and spectral imaging. The sample preparation takes 2 d, equipment setup takes 2-3 h and image acquisition and analysis take 6-8 h.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Broussard JA,Rappaz B,Webb DJ,Brown CMdoi
10.1038/nprot.2012.147subject
Has Abstractpub_date
2013-02-01 00:00:00pages
265-81issue
2eissn
1754-2189issn
1750-2799pii
nprot.2012.147journal_volume
8pub_type
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