EGFP vs monoEGFP - could there be an emission spectrum difference?

[dominika trembecka-lucas]

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Dear Listers,

I have an EGFP monomerised by substitution A206K. It is supposed to be used
for FRET-FLIM measurements. Do you know if this mutation may affect EGFP
fluorescence lifetime even though it is not performed on the chromophore
itself? Have you observed a change in fluorescence lifetime between purified
EGFP and EGFP-6xHisTag?

I will highly appreciate your help.

All the best,

Dominika Trembecka-Lucas

--
Dominika Trembecka - Lucas, MSc
PhD student
Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone] <%2B4812%206646130>

[Vitaly Boyko]

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Hi Dominika,
 
There should not be change in lifetime, though A206K mutation may not be sufficient for GFP being monomeric in in vivo exoerimental setup. Two additional monomeric mutations at positions 221 and 223 could be introduced (not tested though with all three monomeric mutations but described in detail in 2004 Science paper from Roger Tsien lab, if I still remember it right), though these might affect life time, as either 221 or 223 amino acid is located in close proximity to the chromophore. In my hanfds, both CeFP and YFP contained all three monomeric mutation, and it did not affect FRET effciency, though FRET signal  was recorded in very "crowded" envirionment such HIV virus particle, which could accomodate up to 4000 structural proteins tagged with donor (mCeFP) and acceptor (mYFP).
 
Thus, I do not see any reason to worry about. If you are using mRFP1 as acceptor, GFP-mRFP1 may not be the best pair for FRET, I would still favor mCeFP-mYFP (venusYFP did not work well in my hands).
 
Good luck,
 
Vitaly
 


________________________________
From: dominika trembecka-lucas <[hidden email]>
To: [hidden email]
Sent: Thursday, October 20, 2011 6:19 AM
Subject: EGFP vs monoEGFP - could there be an emission spectrum difference?

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To join, leave or search the confocal microscopy listserv, go to:
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Dear Listers,

I have an EGFP monomerised by substitution A206K. It is supposed to be used
for FRET-FLIM measurements. Do you know if this mutation may affect EGFP
fluorescence lifetime even though it is not performed on the chromophore
itself? Have you observed a change in fluorescence lifetime between purified
EGFP and EGFP-6xHisTag?

I will highly appreciate your help.

All the best,

Dominika Trembecka-Lucas

--
Dominika Trembecka - Lucas, MSc
PhD student
Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone] <%2B4812%206646130>

[mcammer]

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Once the EGFP is attached to a another protein, it is likely the lifetime will change.  But does this matter as long as you measure the change which you should do regardless?
-Michael C.

________________________________
From: dominika trembecka-lucas <[hidden email]>
To: [hidden email]
Sent: Thursday, October 20, 2011 6:19 AM
Subject: EGFP vs monoEGFP - could there be an emission spectrum difference?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear Listers,

I have an EGFP monomerised by substitution A206K. It is supposed to be used
for FRET-FLIM measurements. Do you know if this mutation may affect EGFP
fluorescence lifetime even though it is not performed on the chromophore
itself? Have you observed a change in fluorescence lifetime between purified
EGFP and EGFP-6xHisTag?

I will highly appreciate your help.

All the best,

Dominika Trembecka-Lucas

--
Dominika Trembecka - Lucas, MSc
PhD student
Division of Cell Biophysics
Faculty of Biochemistry, Biophysics and
Biotechnology
Jagiellonian University
ul. Gronostajowa 7
30-387 Krakow, Poland
tel. [phone] <%2B4812%206646130>

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[George McNamara]

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6His tag could bring a heavy metal ion close enough to quench.

On 10/20/2011 6:19 AM, dominika trembecka-lucas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Listers,
>
> I have an EGFP monomerised by substitution A206K. It is supposed to be used
> for FRET-FLIM measurements. Do you know if this mutation may affect EGFP
> fluorescence lifetime even though it is not performed on the chromophore
> itself? Have you observed a change in fluorescence lifetime between purified
> EGFP and EGFP-6xHisTag?
>
> I will highly appreciate your help.
>
> All the best,
>
> Dominika Trembecka-Lucas
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami