Fluorescence Resonance Energy Transfer Microscopy As Demonstrated by Measuring the Activation of the Serine/threonine Kinase Akt

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2013 Jan 12

PMID 23306460

Citations 52

Authors

Joshua A Broussard

Benjamin Rappaz

Donna J Webb

Claire M Brown

Affiliations

Soon will be listed here.

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.

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