Introduction to FLIM-FRET techniques

Presented By: David Andrews, PhD

Speaker Biography: Dr. David Andrews is Director of and senior scientist in Biological Sciences at Sunnybrook Research Institute, Professor of Biochemistry and Medical Biophysics at University of Toronto and a Tier 1 Canada Research Chair in Membrane Biogenesis. His research includes, the molecular mechanisms by which Bcl-2 family proteins regulate apoptosis at mitochondria, mechanisms of protein-protein interactions, the assembly of proteins into membranes, high-content screening and development of new microscopes for fluorescence lifetime imaging microscopy.

Webinar: Introduction to FLIM-FRET techniques

Webinar Abstract: Fluorescence Resonance Energy Transfer (FRET) between fluorescence proteins has been implemented for a number of biosensors in which the donor and acceptor are linked in a single sensor. For example, many sensors have been published to measure caspase activity in live cells using a single molecule consisting of a CFP donor linked via a caspase site to an YFP acceptor. In single molecule sensors FRET can be measured using either stimulated emission or Fluorescence Lifetime Imaging Microscopy (FLIM) because the ratio of donor to acceptor is one. However, to measure protein:protein interactions in live cells is more complicated because the relative concentration of donor and acceptor are unknown. Because fluorescence lifetime is independent of concentration, it is possible to use FLIM FRET to quantify binding between any two proteins in live cells. By successfully automating FLIM FRET assays for high throughput we enabled examining the combinatorial interactions between 4 anti-apoptosis proteins with 6 different pro-apoptotic binding partners and their modulation by 15 drugs at 5 concentrations each.