Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt

Joshua A. Broussard, Benjamin Rappaz, Donna J. Webb, Claire M. Brown*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

88 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)265-281
Number of pages17
JournalNature Protocols
Volume8
Issue number2
DOIs
StatePublished - Feb 2013

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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