The Huck Institutes of the Life Sciences

FRET with confocal microscopy

An example of using fluorescence resonance energy transfer (FRET) to study protein localization, dynamics and interactions in cells


What is ?

(Fluorescence Resonance Energy Transfer) is one of a few methods that can determine if molecules are less than 10nm apart:

  • A donor fluorochrome is attached to one protein
  • An acceptor fluorochrome is attached to another protein
  • If the distance between proteins is close enough, an excited donor will transfer its energy to the acceptor, causing it to fluoresce under donor excitation conditions

One of the most common FRET pairs is CFP and YFP. These variants of GFP, green fluorescent protein, have revolutionized the ability to study protein localization, dynamics and interactions in cells.

Our FRET capabilities

Both of the Facility's confocal microscopes are fitted with CFP-YFP configurations, as well as many other FRET pairs.

Calculation of FRET efficiencies can be done with the FV1000 software(it has a PFRET algorithm) or with our PFRET program.

FRET example

Lorraine Santy, a faculty member in the Department of Biochemistry and Molecular Biology, has used our confocal and widefield microscopes to show CFP-YFP interactions in the Golgi apparatus. The small GTPase ARF1 regulates trafficking in the Golgi apparatus. Hela cells were transfected with ARF1-CFP and GGA1-YFP. GGA1 is a Golgi effector protein that binds only to the activated form of ARF1. Therefore, the FRET interaction indicates the presence of active ARF1.