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
2013 Nature Protocols  
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
more » ... ivation. 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.
doi:10.1038/nprot.2012.147 pmid:23306460 pmcid:PMC3756929 fatcat:6qld5ibsurha3hwhiv6ntp44oa