Posted by
Maria Calvo-2 on
Apr 07, 2010; 4:14pm
URL: http://confocal-microscopy-list.275.s1.nabble.com/Best-FRET-pair-tp4804703p4865853.html
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
>
--
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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]
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