Hi Simon,

It all depends on the application and the final goals.
If you are working under conditions of high overexpression, that it doesn't 
matter much. However, if you would be using weak promoters, create cell lines 
expressing a fusion protein of interest, then many things become important. 

For example, I would use CeFP fusion protein as a reference or as a fusion to 
relatively abundant proteins in the cell (e.g. actin, tubulin, histones, etc).

YFP channel is very good, in relation to SNR, but I may suggest a fusion to 
tandem, 2x_EYFP or 2x_venusYFP (with one or all three monomeric mutations). For 
the red channel mCherry (or mRFP1 fused to the N- and C-terminal peptides of 
GFP origin), like in mCherry. If your expression vectors are of viral origin, 
or if you may suspect aberrant splicing artefacts in your model system, then 
M10L version of mCherry will solve the problem (i.e. contamination by free, 
unfused mCherry).

Autofluorescence is a serious obstacle in low light microscopy, thus I would 
recommend adapting your cell cultures to OPTI-MEM media with 2-3% FCS.

Many others proteins are either less soluble or di- through tetramers, may 
suffer from the low quantum yield, low SNR, etc.

Recently, there were few papers on new FPs which sounded interesting, but 
nothing alike "supersonic".... 
mCherry, especially in its 2x tandem variant is still a "trouble-maker" (has a 
tendency to form aggregates as fusion to a number of proteins tested myself in 
vivo), and I doubt that mCherry is monomeric under conditions of 
overexpression - in a simple BiFC experiment where mCherry was fused to split 
YFP (mCherry_ny and mCherry-cy), YFP signal reconstitution is as 
quick_and_strong as in the positive control (CD8_ny plus CD8-cy; and CD8 is 
known to live as a dimer).

If you have any further questions, please contact me off-line. 

Vitaly
NCI-Frederick,
301-846-6575
  


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On 
Behalf Of Simon Walker
Sent: Friday, July 31, 2009 10:54 AM
To: [log in to unmask]
Subject: Fluorescent proteins

Dear all,
One common question I get is "What is the best combination of fluorescent 
proteins to use in my live cell imaging experiment?".  Based on experience my 
advice for a double labelling experiment would be Cerulean and mVenus, or 
perhaps EGFP and mCherry and for a triple labelling experiment Cerulean, mVenus 
and mCherry.  
It seems that every few months a new contender to the best FP crown appears, 
and I was just wondering if anyone has any 'real world' experience of some of 
these newer variants which would lead them to chose a different combination 
from the ones listed above.  For example, has anyone successfully used Azurite, 
mTeal, Emerald, YPet, TagRFP or mKate either in isolation or in combination?  
Does anyone have bad experiences of these FPs?
Thanks,
Simon
Babraham Institute, UK


-------------------------------------------------