Donna Webb
Last active: 2/5/2016

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

Broussard JA, Rappaz B, Webb DJ, Brown CM
Nat Protoc. 2013 8 (2): 265-81

PMID: 23306460 · PMCID: PMC3756929 · DOI:10.1038/nprot.2012.147

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.

MeSH Terms (9)

Enzyme Activation Fluorescence Polarization Fluorescence Resonance Energy Transfer Image Processing, Computer-Assisted Microscopy Models, Molecular Optical Imaging Photobleaching Proto-Oncogene Proteins c-akt

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