A Beginner’s Guide to the Principles and Applications of FRET

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Welcome to our sixth webinar
A Beginner’s Guide to the Principles and
Applications of FRET
Dr Andy Lane

Dr Andy Lane
• FRET background and key
considerations in experimental design
• Examples of applications of FRET
• Labelling of reagents for FRET

FRET background
FRET, or Fluorescence Resonance Energy Transfer, was first described more than
50 years ago. The availability of new dyes and detection technology has resulted in
a much wider use of the application in recent years, especially in biomedical R&D.
In simple terms FRET is dependent on the distance dependent transfer of
energy from a donor molecule to an acceptor molecule, resulting in the loss of
fluorescence of the donor and gain of fluorescence of the acceptor – either of
which may be measured.
FRET is therefore an extremely powerful tool for identifying molecular interactions. It can
be used in a variety of base techniques, including microscopy, flow cytometry and ELISA.

Conditions influencing FRET
 DISTANCE – donor and acceptor molecules must be in close proximity to each
other, generally between 10-100 Å
 SPECTRA – the absorption spectrum of the acceptor must overlap the emission
spectrum of the donor
 DIPOLE ORIENTATION – donor and acceptor molecule dipole orientations should
be approximately parallel

Conditions influencing FRET - distance
Assuming other criteria are met, the most important factor is the distance between
the pair of molecules.
Förster showed that the efficiency (E) of FRET is dependent on the inverse sixth-distance
between the pair, where E = Ro6 / (Ro6 + r6). Ro is known as the Förster distance,
being that at which 50% of the energy is transferred, and r is the actual distance
between the pair.
In practical terms this means that the efficiency of FRET is extremely sensitive to distance
– if the molecules are moved 10x further apart, the efficiency falls 5million fold.

Conditions influencing FRET - spectra
As in the diagram below the emission and absorption spectra of the donor/acceptor pair
must overlap. The degree of overlap has an impact, amongst other factors, on the
Förster distance.
Wavelength
Emission
(donor)
Absorption
(acceptor)
Absorption
(donor)
Emission
(acceptor)
Normalisedintensity

Measurement of FRET
FRET is effectively measured in one of two ways:-
1. The increase in fluorescence of the acceptor molecule
2. The decrease in fluorescence of the donor molecule
There may also be advantages to expressing this as a ratio, which means that
the measurement is then not dependent on the physical concentration
of the molecules.
“Acceptor photobleaching” is a widely used variant, whereby bleaching of the
acceptor molecule leads to an increase in donor emission, showing that FRET
was occurring in the system.

Commonly used FRET pairs
DONOR ACCEPTOR
Fluorescein TRITC
CFP YFP
BFP GFP
Europium APC / DyLight® 650
Terbium RPE / DyLight® 550
DyLight® 488 DyLight® 550
Cy3 Cy5

FRET experimental design (1)
Selection of appropriate donor / acceptor pairs based both on their inherent
suitability as a pair, and your equipment.
Choose suitable filters to maximise excitation of your donor, whilst minimising
any direct excitation of the acceptor molecule.
Consider concentration – are there likely to be enough detectable events?
High concentrations of both donor and acceptor might not be enough to generate
a signal if the interaction is very low – remember that it is the interaction
that is being measured.

FRET experimental design (2)
Consider which molecule might be better as the donor and which the acceptor.
Unpaired donor molecules dilute a signal, whilst unpaired acceptors have very little
Effect. If possible use the molecule at lowest level as the donor.
If possible use a control for the FRET reaction itself. A simple control is the use of a
primary antibody labelled as an acceptor and a secondary antibody labelled as a donor.
All donor labelled antibodies will be adjacent to acceptors, giving high efficiency FRET.

FRET examples
Typical example of detecting interaction of two molecules – e.g. drug binding to active site
or antibody binding to target

Blocking of
biological interaction
Donor emissionAcceptor emission
FRET examples
Example of detecting a compound that blocks interaction of two molecules

FRET examples
Detecting co-location of molecules on/within cells

FRET examples
Example of protein cleavage detected by loss of FRET signal

FRET examples
Example of detection
of conformational
change
FRET

FRET examples
Construction of tandem dyes for flow cytometry
RPE
Cy7
Cy7
Cy7
Cy7
RPE
488nm
775nm
575nm
488nm

FRET reagent preparation
 Genetically encoded dye incorporation – e.g. GFP, YFP etc
 Chemical conjugation of target molecules – e.g. antibodies,
other proteins, oligonucleotides
Note that there may be advantages to using antibodies against specific
target molecules if they are available rather than using genetic encoding.

• Lightning-Link ® - the world’s easiest protein labelling kits
• Simple, one step process
• Only 30 seconds hands-on
• Reproducible
• Scalable µg to mg
• 100% recovery
Just add primary antibody !
19

Lightning-Link®

Lightning-Link® Rapid
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

Conjugation considerations
You need to know some things about your reagent.
Lightning-Link conjugations are really simple
but you need protein in the right format to work effectively.
Concentration – 1mg/ml or higher is preferred
Purity – ensure other proteins have been removed,
and also make sure they haven’t been put back again afterwards!
Buffer formulation – most common formulations are suitable,
but ensure that amines such as glycine are truly absent,
as well as thiols such as DTT or mercaptoethanol. Tris is OK up to 20mM
Lightning Link kits are optimised for antibody labelling, but can easily be
adjusted to label other proteins. If you know the size of your protein you can
calculate how to use Lightning Link for your application

Contact
If you would like any more information, please contact us at
info@innovabiosciences.com
Please keep an eye out for our future webinars and other exciting news on our
website and social media channels:
www.innovabiosciences.com/innova/webinars.html
YouTube: www.youtube.com/InnovaBiosciences

Innova Biosciences Ltd.
Babraham Research Campus,
Cambridge, UK,
CB22 3AT
www.innovabiosciences.com
Lightning-Link® is a registered trademark of Innova Biosciences
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries

A Beginner’s Guide to the Principles and Applications of FRET

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