| A High Content FRET Assay by Two-Color Anisotropy |
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Presentation: P02003 Session: Imaging Technologies - Poster SessionEvan F. Cromwell, Madeleine L. Craske, Mark A. Rizzo (Vanderbilt University), David W. Piston (Vanderbilt University), Steven C. Miller, Blueshift Biotechnologies, Inc. Presenting Author: Evan Cromwell, Blueshift Biotechnologies - USA Detection of Förster resonance energy transfer (FRET) between fluorescent proteins holds great promise for studying live-cell protein-protein interactions. Difficulties in quantitation through donor-acceptor emission ratios have limited the use of FRET in high content screening. A new strategy for detecting FRET based on depolarized sensitized emission has recently been developed. In this method, anisotropy of the acceptor emission changes from a high value in the absence of FRET to a lower value in the presence of energy transfer. Here we report on the use of this technique with the IsoCyte™ laser scanning fluorimeter to quantify FRET with a CFP-YFP donor-acceptor pair. Comparison is made between emission ratio and acceptor anisotropy methods using both CFP-YFP calibration beads and cells transfected with CFP, YFP, and FRET constructs. Results from a live-cell caspase-3 activation assay will be presented. |