Posted by
yuansheng sun on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Best-FRET-pair-tp4804703p4871864.html
Hi Maria,
This is sheng, working for Ammasi. From the pair list, I would
recommend A488-mOrange, at least for sensitized FRET, assuming you
have optimal Ex sources and Em filters. I did a quick calculation of
the Ro for this pair - 5.889 nm, based on the following photophysical
properties of the two fluorophores:
Alexa 488 (Invitrogen T13342): QY = 0.92 (Be aware that the Alexa QY
will likely drop down a lot when it is conjugated)
mOrange: QY = 0.69, EC = 71,000, Brightness to EGFP = 146%.
I can send you the spectra of the two fluorophores if you want.
The photostability of mOrange is very poor and this might be a good
thing for apFRET. However, it has been reported that mOrange may have
a photoconversion issue (Nat.Methods, 2009, 6, 5, 355-358). Please do
your controls carefully. If you use lasers in sensitized FRET, I do
not suggest the DPSS 561 nm laser for the acceptor excitation, because
it will limit the detection range of your FRET channel.
To be honest, I have no experience on the A488-mOrange pair. Based on
the paper, it should work. Another pair to try would be YFP-A555.
sheng
On Wed, Apr 7, 2010 at 12:14 PM, Maria Calvo <
[hidden email]> wrote:
> Dear all,
> (and specially Ammasy and Vitaly, who answered my previous mail),
>
> Sorry if I didn't explain it well or I missed some details.
>
> We want to detect FRET by combining a fluorescent protein and a directly
> labelled primary antibody. The methods we are going to use are: sensitized
> emission and/or acceptor photobleaching (not lifetime imaging).
>
> 1) Fluorescent Proteins: We have only available the fusion protein
> constructs with YFP, mCherry or mOrange.
> 2) Fluorochromes: We will buy a kit to labell the primary antibody.
>
> We have to decide which fluorophore will be the best in combination with one
> of these fluorescent proteins.
>
> The combinations I thought were:
>
> -Donor - Acceptor
>
> -YFP -Alexa 594 or TR
> -YFP - A555 or Cy3
> -mCherry - Cy5 or A647
> -mOrange - Cy5 or A647
> -FITC or A488 - mOrange
> -FITC or A488 - mCherry
>
> Which do you think is the best combination in terms of J (overlap),
> brightness?
>
> Thank you for your invaluable help!
>
> Maria Calvo
>
>
>
> Maria Calvo escribió:
>>
>> Dear all,
>>
>> We are planning to do FRET experiments with a fluorescent protein (YFP,
>> mCherry or mOrange)and a directly labelled primary antibody.
>> We are restricted to YFP, mCherry or mOrange for one of the proteins (the
>> most abundant).
>> Does anyone know what's the best FRET pair for any of these FPs, in terms
>> of J (overlap), brightness ?
>>
>> Thanks for your help,
>>
>> Maria Calvo
>>
>
>
> --
>
>
> ___________________________________
>
> Dra. Maria Calvo
>
> Unitat de Microscòpia Confocal
> Serveis Cientificotècnics-C.Casanova
> Facultat de Medicina
> Universitat de Barcelona- IDIBAPS
> C/ Casanova 143
> Barcelona 08036
>
> Tel: 34 934037159/39930
> Fax: 34 934039946
> E-mail:
[hidden email]
> ___________________________________
>