photoconvertible red/green proteins-quest for experience

[Ekaterina Papusheva]

Hi,

I have a question about tagging proteins with photoconvertoble fluorophores.

We are thinking about using one of the photoconvertible fluorescent proteins
(Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the protein of
interest and 2-channel imaging (red/green).

We would like to know which one is a best choice - ie, folding fine in if
inserted in the middle of a protein, bright and stabe enough for
time-lapse confocal imaging (about 30 time points with 20 z-planes each),
photoconversion not too difficult.... any other considerations that might
occur to you are also wellcome.

We would really appreciate your help,

Best regards,
Ekaterina


--
postdoc Heisenberg lab
Max-Planck-Institute for Molecular Cell Biology and Genetics
Pfotenhauerstr.108
D-01307 Dresden
Germany
fon (lab) [phone]

[David Stanek-3]

Hi Ekaterina,

We used Dendra2 tagged histones in HeLas. Photoconversion as well as time-lapse (20h every 30min) was fine and protein was stable in both channels (see Cvackova et al. JSB, 2008).  However, tagging of some other nuclear proteins was not that successful and Dendra2 behaved unpredictably (e.g. no photoconversion or increased green fluorescence instead of photoconversion). 

David

David Stanek, PhD.

Department of RNA biology

Institute of Molecular Genetics AS CR

Videnska 1083

142 20 Prague 4

Czech Republic

Tel.: [phone]

Fax: [phone]

email: [hidden email]

web: www.img.cas.cz

On 08 Dec 2009, at 15:14, Ekaterina Papusheva wrote:

Hi,

I have a question about tagging proteins with photoconvertoble fluorophores.

We are thinking about using one of the photoconvertible fluorescent proteins

(Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the protein of

interest and 2-channel imaging (red/green).

We would like to know which one is a best choice - ie, folding fine in if

inserted in the middle of a protein, bright and stabe enough for

time-lapse confocal imaging (about 30 time points with 20 z-planes each),

photoconversion not too difficult.... any other considerations that might

occur to you are also wellcome.

We would really appreciate your help,

Best regards,

Ekaterina

--

postdoc Heisenberg lab

Max-Planck-Institute for Molecular Cell Biology and Genetics

Pfotenhauerstr.108

D-01307 Dresden

Germany

fon (lab) [phone]

[Ekaterina Papusheva]

Thanks a lot for he feedback!

I really appreciate it,
Ekaterina

> Hi Ekaterina,
>
> We used Dendra2 tagged histones in HeLas. Photoconversion as well as
> time-lapse (20h every 30min) was fine and protein was stable in both
> channels (see Cvackova et al. JSB, 2008).  However, tagging of some
> other nuclear proteins was not that successful and Dendra2 behaved
> unpredictably (e.g. no photoconversion or increased green
> fluorescence instead of photoconversion).
>
> David
>
> David Stanek, PhD.
> Department of RNA biology
> Institute of Molecular Genetics AS CR
> Videnska 1083
> 142 20 Prague 4
> Czech Republic
> Tel.: [phone]
> Fax: [phone]
> email: [hidden email]
> web: www.img.cas.cz
>
> On 08 Dec 2009, at 15:14, Ekaterina Papusheva wrote:
>
>> Hi,
>>
>> I have a question about tagging proteins with photoconvertoble
>> fluorophores.
>>
>> We are thinking about using one of the photoconvertible fluorescent
>> proteins
>> (Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the
>> protein of
>> interest and 2-channel imaging (red/green).
>>
>> We would like to know which one is a best choice - ie, folding fine
>> in if
>> inserted in the middle of a protein, bright and stabe enough for
>> time-lapse confocal imaging (about 30 time points with 20 z-planes
>> each),
>> photoconversion not too difficult.... any other considerations that
>> might
>> occur to you are also wellcome.
>>
>> We would really appreciate your help,
>>
>> Best regards,
>> Ekaterina
>>
>>
>> --
>> postdoc Heisenberg lab
>> Max-Planck-Institute for Molecular Cell Biology and Genetics
>> Pfotenhauerstr.108
>> D-01307 Dresden
>> Germany
>> fon (lab) [phone]
>
>
>
>
>
>
>
>

--
postdoc Heisenberg lab
Max-Planck-Institute for Molecular Cell Biology and Genetics
Pfotenhauerstr.108
D-01307 Dresden
Germany
fon (lab) [phone]

[Michael Schell]

We fused Lifeact to Dendra2, and that worked well.   It switched very nicely, especially with the Olympus "tornado bleach." Some other F-actin side-binding protein domains aggregated more when fused to Dendra2 than they did with EGFP.  But the performance of Dendra2 was as good as any RFP we've tried (we have not tried mApple yet).  

One thing we did note for Dendra2:  prior to photo-switching, the green fluorescence seems to go through a stage when it gets brighter than the starting fluorescence.  When conversion occurs, the green then diminishes as expected.

Mike



Michael J. Schell, Ph.D., CIV, USUHS
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  [phone]
[hidden email]
>>> David Stanek <[hidden email]> 12/08/09 9:38 AM >>>
Hi Ekaterina,

We used Dendra2 tagged histones in HeLas. Photoconversion as well as  
time-lapse (20h every 30min) was fine and protein was stable in both  
channels (see Cvackova et al. JSB, 2008).  However, tagging of some  
other nuclear proteins was not that successful and Dendra2 behaved  
unpredictably (e.g. no photoconversion or increased green  
fluorescence instead of photoconversion).

David

David Stanek, PhD.
Department of RNA biology
Institute of Molecular Genetics AS CR
Videnska 1083
142 20 Prague 4
Czech Republic
Tel.: [phone]
Fax: [phone]
email: [hidden email]
web: www.img.cas.cz

On 08 Dec 2009, at 15:14, Ekaterina Papusheva wrote:

> Hi,
>
> I have a question about tagging proteins with photoconvertoble  
> fluorophores.
>
> We are thinking about using one of the photoconvertible fluorescent  
> proteins
> (Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the  
> protein of
> interest and 2-channel imaging (red/green).
>
> We would like to know which one is a best choice - ie, folding fine  
> in if
> inserted in the middle of a protein, bright and stabe enough for
> time-lapse confocal imaging (about 30 time points with 20 z-planes  
> each),
> photoconversion not too difficult.... any other considerations that  
> might
> occur to you are also wellcome.
>
> We would really appreciate your help,
>
> Best regards,
> Ekaterina
>
>
> --
> postdoc Heisenberg lab
> Max-Planck-Institute for Molecular Cell Biology and Genetics
> Pfotenhauerstr.108
> D-01307 Dresden
> Germany
> fon (lab) [phone]

Classification:  UNCLASSIFIED
Caveats: FOUO

[Stefan Terjung]

> One thing we did note for Dendra2:  prior to photo-switching, the green
> fluorescence seems to go through a stage when it gets brighter than the
> starting fluorescence.  When conversion occurs, the green then diminishes as
> expected.

We also made the observation that Dendra2 seems to go through a stage when it gets brighter. Maybe this is due to the sequential absorption of 2 photons for the photoconversion Gurskaya et al. 2006, Nature Biotechnology 24(4): 461-465 describe:
"We observed a nonlinear, sigmoidal dependence (Supplementary Fig. 3b online) indicating that Dendra requires sequential absorption of two photons to be converted into the red state."

To me this brighter green state seemed more pronounced in fast moving proteins compared to rather "immobilized" proteins.

------------------------
Dr. Stefan Terjung
Advanced Light Microscopy Facility
EMBL Heidelberg
Meyerhofstr.1
D - 69117 Heidelberg

>
> Mike
>
>
>
> Michael J. Schell, Ph.D., CIV, USUHS
> Assist. Professor
> Dept. of Pharmacology
> Uniformed Services University
> 4301 Jones Bridge Rd.
> Bethesda, MD  20814-3220
> tel:  [phone]
> [hidden email]
> >>> David Stanek <[hidden email]> 12/08/09 9:38 AM >>>
> Hi Ekaterina,
>
> We used Dendra2 tagged histones in HeLas. Photoconversion as well as  
> time-lapse (20h every 30min) was fine and protein was stable in both  
> channels (see Cvackova et al. JSB, 2008).  However, tagging of some  
> other nuclear proteins was not that successful and Dendra2 behaved  
> unpredictably (e.g. no photoconversion or increased green  
> fluorescence instead of photoconversion).
>
> David
>
> David Stanek, PhD.
> Department of RNA biology
> Institute of Molecular Genetics AS CR
> Videnska 1083
> 142 20 Prague 4
> Czech Republic
> Tel.: [phone]
> Fax: [phone]
> email: [hidden email]
> web: www.img.cas.cz
>
> On 08 Dec 2009, at 15:14, Ekaterina Papusheva wrote:
>
> > Hi,
> >
> > I have a question about tagging proteins with photoconvertoble  
> > fluorophores.
> >
> > We are thinking about using one of the photoconvertible fluorescent  
> > proteins
> > (Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the  
> > protein of
> > interest and 2-channel imaging (red/green).
> >
> > We would like to know which one is a best choice - ie, folding fine  
> > in if
> > inserted in the middle of a protein, bright and stabe enough for
> > time-lapse confocal imaging (about 30 time points with 20 z-planes  
> > each),
> > photoconversion not too difficult.... any other considerations that  
> > might
> > occur to you are also wellcome.
> >
> > We would really appreciate your help,
> >
> > Best regards,
> > Ekaterina
> >
> >
> > --
> > postdoc Heisenberg lab
> > Max-Planck-Institute for Molecular Cell Biology and Genetics
> > Pfotenhauerstr.108
> > D-01307 Dresden
> > Germany
> > fon (lab) [phone]
>
>
>
>
>
>
>
>
> Classification:  UNCLASSIFIED
> Caveats: FOUO

--
-------------------------------------------------------
Stefan Terjung             [hidden email]
Am Kirchberg 3            Tel.  [phone]
69221 Dossenheim

Preisknaller: GMX DSL Flatrate für nur 16,99 Euro/mtl.!
http://portal.gmx.net/de/go/dsl02

[Roland Nitschke]

We observed the same behaviour, strong increase of the green signal ( 2
- 4 x brighter) before conversion, independent of using 351, 364 , 405
or 2-P for conversion. With green 488 we only had poor results compared
to UV or 2-P. Also one has to be quite careful of adjusting the
conversion intensity to avoid going more or or less directly into
bleaching.

Roland

Stefan Terjung schrieb:

>> One thing we did note for Dendra2:  prior to photo-switching, the green
>> fluorescence seems to go through a stage when it gets brighter than the
>> starting fluorescence.  When conversion occurs, the green then diminishes as
>> expected.
>>    
>
> We also made the observation that Dendra2 seems to go through a stage when it gets brighter. Maybe this is due to the sequential absorption of 2 photons for the photoconversion Gurskaya et al. 2006, Nature Biotechnology 24(4): 461-465 describe:
> "We observed a nonlinear, sigmoidal dependence (Supplementary Fig. 3b online) indicating that Dendra requires sequential absorption of two photons to be converted into the red state."
>
> To me this brighter green state seemed more pronounced in fast moving proteins compared to rather "immobilized" proteins.
>
> ------------------------
> Dr. Stefan Terjung
> Advanced Light Microscopy Facility
> EMBL Heidelberg
> Meyerhofstr.1
> D - 69117 Heidelberg
>
>  
>> Mike
>>
>>
>>
>> Michael J. Schell, Ph.D., CIV, USUHS
>> Assist. Professor
>> Dept. of Pharmacology
>> Uniformed Services University
>> 4301 Jones Bridge Rd.
>> Bethesda, MD  20814-3220
>> tel:  [phone]
>> [hidden email]
>>    
>>>>> David Stanek <[hidden email]> 12/08/09 9:38 AM >>>
>>>>>          
>> Hi Ekaterina,
>>
>> We used Dendra2 tagged histones in HeLas. Photoconversion as well as  
>> time-lapse (20h every 30min) was fine and protein was stable in both  
>> channels (see Cvackova et al. JSB, 2008).  However, tagging of some  
>> other nuclear proteins was not that successful and Dendra2 behaved  
>> unpredictably (e.g. no photoconversion or increased green  
>> fluorescence instead of photoconversion).
>>
>> David
>>
>> David Stanek, PhD.
>> Department of RNA biology
>> Institute of Molecular Genetics AS CR
>> Videnska 1083
>> 142 20 Prague 4
>> Czech Republic
>> Tel.: [phone]
>> Fax: [phone]
>> email: [hidden email]
>> web: www.img.cas.cz
>>
>> On 08 Dec 2009, at 15:14, Ekaterina Papusheva wrote:
>>
>>    
>>> Hi,
>>>
>>> I have a question about tagging proteins with photoconvertoble  
>>> fluorophores.
>>>
>>> We are thinking about using one of the photoconvertible fluorescent  
>>> proteins
>>> (Dendra2, dEosFP-Tandem or mEos2FP) for internal fusion in the  
>>> protein of
>>> interest and 2-channel imaging (red/green).
>>>
>>> We would like to know which one is a best choice - ie, folding fine  
>>> in if
>>> inserted in the middle of a protein, bright and stabe enough for
>>> time-lapse confocal imaging (about 30 time points with 20 z-planes  
>>> each),
>>> photoconversion not too difficult.... any other considerations that  
>>> might
>>> occur to you are also wellcome.
>>>
>>> We would really appreciate your help,
>>>
>>> Best regards,
>>> Ekaterina
>>>
>>>
>>> --
>>> postdoc Heisenberg lab
>>> Max-Planck-Institute for Molecular Cell Biology and Genetics
>>> Pfotenhauerstr.108
>>> D-01307 Dresden
>>> Germany
>>> fon (lab) [phone]
>>>      
>>
>>
>>
>>
>>
>>
>> Classification:  UNCLASSIFIED
>> Caveats: FOUO
>>    
>
>  

--
___________________________
Nitschke, Roland Dr.
Life Imaging Center
Centre of Systems Biology
Albert-Ludwigs-University Freiburg
Habsburgerstr.49
D-79104 Freiburg
Germany
___________________________
E-mail: [hidden email]
phone: [phone] or 2902
fax: [phone]
http://www.imaging.uni-freiburg.de
http://zbsa.de