Introduction to FLIM-FRET techniques

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.

In this webinar we will discuss the benefits and limitations of different methods for measuring FRET in cells. I will describe the exceptional utility of this kind of data in early stage drug development. Also discussed will be the steps required to surmount the challenges posed when generating 1800 binding curves in a single experiment with live cells and how to interpret the data to provide the most useful single value to guide medicinal chemistry efforts. Finally, we will explore some of the unexpected insights garnered from our data examining inhibitors on anti-apoptosis proteins in live cells.  

Learning Objectives:

1. Understanding what FRET is and how it can be used to measure protein:protein interactions

2. Understanding how FRET can be measured in live cells using automated microscopy