FRET

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Cynthia
Cynthia's picture
FRET

Is FRET always performed with CFP-YFP tags? Why is this?

Guy Sovak
Guy Sovak's picture
Hi Cynthia,

Hi Cynthia,
The answer is not always.
Please look at this link and you will find a good description.
http://probes.invitrogen.com/handbook/boxes/0422.html
Guy

Fraser Moss
Fraser Moss's picture
No

No

all you need is a suitable pair for which the emission spectra of the donor overlaps sufficiently with the excitation spectra of the acceptor to get FRET.

e.g. FITC & TRITC, Cy3 & Cy5, eGFP & Cy3

here's a good teaching guide to get you started

FRET teaching aid

another cool tutorial can be found at

http://www.microscopyu.com/tutorials/java/fluorescence/fpfret/index.html

Guy Sovak
Guy Sovak's picture
Thanks Frasermoss for the

Thanks Frasermoss for the nice FRET teaching ade module.
Very interesting and informative.
Guy

Cynthia
Cynthia's picture
Thanks for the assistance.

Thanks for the assistance. This has been very helpful as we initiate FRET experiments. Cynthia

JUAN ANTONIO
JUAN ANTONIO's picture
 Hi,

 Hi,
I have another question related to FRET.
How important is the quantum yield of the donor in the process?
Could I develop a  FRET pair with TAMRA or a similar fluorophore as aceptor, where the donor has a quantum yield of 0.2? 
Thanks

Fraser Moss
Fraser Moss's picture
The quantum yield of the

The quantum yield of the donor is important.

First some backgroud.

For those reading this post who do not know what Quantum yield is, it is the ratio between the number of photons emitted by the fluorophore to the number of photon absorbed.

In a Forster resonance energy transfer (FRET) experiment, the quantum yield of the donor and acceptor fluorophore are written as QD and QA respectively.  QDA is the quantum yeild of the donor on the presence of the acceptor.

The energy absorbed by an excited fluorophore must underdo decay in order for it to return to the ground state, and it achieves this through both radiative (fluorescence emission) and non-radiative decay

The smaller the QD of the donor, the more photon energy will dissipated via non-radiative decay compared to the radiative fluorecent emission of the donor, or possible FRET.

The proportion of the photon energy absorbed by the donor that can be transferred to the acceptor without fluorescence emission is represented by the FRET efficiency (E).

The relationship between E and Q in a FRET experiment is represented by

 QDA/QD +E = 1

QDA/QD is the efficiency of the radiative (fluorescent) process.

Now in your case, you are considering a FRET partner for TAMRA with TAMRA as the acceptor (unusual!).  If the donor has QD of 0.2, then you can use this in the calculation of R0, the Forster distance for the donor-acceptor pair.

R06 = 8.79 x 1023 k2 n-4 QD J

8.79 x 1023 = Avagadro constant
k2 = dipole orientation factor (assumed to be 2/3 for most systems)
n = the referactive index of the medium
J = the spectral overlap integral

For donors having a high quantum yield and acceptors with large extinction coefficients, the spectral overlap integral will be greater, leading to more efficient FRET partners with a higher Ro value. 

where FD(λ) is the fluorescence intensity of the energy donor at wavelength λ (in cm), and ε(λ) is the molar  extinction coefficient (in cm-1 M-1) of the acceptor.

If you know how to use Matlab, somone has actually written a routine to help you calulate J and R0 if you know the ε(λ) and QD

TAMRA has an extinction coefficent (580nm) of 91,000 cm-1 M-1

The size if R0 obviousy effects the sensitivity of any measurements of fluorophore separation you intend to make with your donor- and acceptor-tagged molecules

R = R0 (E-1 -1)1/6

All that said, a QD of 0.2 is very small and I'd be concerned that you would not be able to detect any FRET occuring in your system (signal to noise may not large enough).  What is the extinction coefficient of this donor and does its fluorescence lifetime decay with a single exponential or more than one exponential?

See Rizzo et al Nat Biotechnol. 2004 Apr;22(4):445-9 for background on optimizing donors for FRET experiments.

JUAN ANTONIO
JUAN ANTONIO's picture
 Hi Frassermoss,

 Hi Frassermoss,

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line-height:150%">
mso-ansi-language:EN-US">Thanks for your quick response.

line-height:150%">
mso-ansi-language:EN-US">My name is Juan Antonio and I am working at Scripps as Research Scientist.

line-height:150%">
mso-ansi-language:EN-US">I am telling you what I want to do. I have a fluorescent peptide sensor of 12 amino acids, wearing a fluorophore (F1) and all what I want to do is to improve the emission wavelength of this sensor using FRET. The fluorophore attached to the peptide, fluorophore F1 (
mso-ansi-language:EN-US">l
line-height:150%;mso-ansi-language:EN-US">ex = 365 nm and
mso-ansi-language:EN-US">l
line-height:150%;mso-ansi-language:EN-US">em = 485 nm), has an extinction coefficient of 8,000 cm-1 M-1 and a quantum yield of 0.34. I would like to know if it would be possible to find a proper acceptor (fluorophore F2) for fluorophore F1 in order to make FRET.

line-height:150%">
mso-ansi-language:EN-US">Alternatively I have another sensor where I would also like to make FRET. In this peptide sensor the fluorophore F1 (
mso-ansi-language:EN-US">l
line-height:150%;mso-ansi-language:EN-US">ex = 375 nm and
mso-ansi-language:EN-US">l
line-height:150%;mso-ansi-language:EN-US">em = 525 nm), has an extinction coefficient of 7,900 cm-1 M-1 and a quantum yield of 0.12. I would like also like to know if it would be possible to find a proper acceptor (fluorophore F2) for fluorophore F1 in order to make FRET.

line-height:150%">
mso-ansi-language:EN-US"> 

line-height:150%">
mso-ansi-language:EN-US">Thank you very much, you are helping me a lot,

line-height:150%">
mso-ansi-language:EN-US"> 

line-height:150%">
mso-ansi-language:EN-US">Juan Antonio
 
 
 

JUAN ANTONIO
JUAN ANTONIO's picture
 I would like to know if I

 I would like to know if I can stimate the Förster distance for a new FRET pair using photochemcad. I have the emision spectrum of the donor normalized to unit. Can I use the absorcion spectra of the fluorophores of the Photochemcad data base to stimate Ro? Thanks

Vira
Vira's picture
Respected Scientists,

Respected Scientists,

I wish to do somework in FRET using Confocal Microscope. I understand that I can also use dye solutions like organic laser donor-acceptor dyes to study FRET in confocal microscope. It is not neccessary that we should use FRET only for biological samples, right?

Can anyone suggest to me about what are the different ways in which I can approach this topic to find out the best donor-acceptor pair which gives max. FRET efficiency (from the pairs I may choose)? For e.g., for a common acceptor, I can use different donors to find out which donor when used with that acceptor will give good FRET efficiency. Then this choice of the best donor may depend on what factors? is it structure of the donor, or maybe quantum yield of the donor (as one scientist as explained in the previous posts), or etc.? can anyone suggest to me about these different parameters that may influence the fret effeciency.